Next Article in Journal
AKVANO®: A Novel Lipid Formulation System for Topical Drug Delivery—In Vitro Studies
Previous Article in Journal
Daunorubicin and Its Active Metabolite Pharmacokinetic Profiles in Acute Myeloid Leukaemia Patients: A Pharmacokinetic Ancillary Study of the BIG-1 Trial
Previous Article in Special Issue
In Vivo Investigation of (2-Hydroxypropyl)-β-cyclodextrin-Based Formulation of Spironolactone in Aqueous Solution for Paediatric Use
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies

The Molecular Genetics Thalassemia Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus
Empa, Swiss Federal Laboratories for Materials Science and Technology, 9014 St. Gallen, Switzerland
Institute of Translational Pharmacology, IFT National Research Council, 90146 Palermo, Italy
Pediatric Clinic, Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, Fondazione IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy
Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, 50005 Hradec Králové, Czech Republic
Consorzio per Valutazioni Biologiche e Farmacologiche (CVBF) and European Paediatric Translational Research Infrastructure (EPTRI), 70122 Bari, Italy
Author to whom correspondence should be addressed.
Pharmaceutics 2022, 14(4), 793;
Submission received: 28 February 2022 / Revised: 29 March 2022 / Accepted: 1 April 2022 / Published: 5 April 2022


Advanced therapy medicinal products (ATMPs) are medicines for human use based on genes, cells or tissue engineering. After clear successes in adults, the nascent technology now sees increasing pediatric application. For many still untreatable disorders with pre- or perinatal onset, timely intervention is simply indispensable; thus, prenatal and pediatric applications of ATMPs hold great promise for curative treatments. Moreover, for most inherited disorders, early ATMP application may substantially improve efficiency, economy and accessibility compared with application in adults. Vindicating this notion, initial data for cell-based ATMPs show better cell yields, success rates and corrections of disease parameters for younger patients, in addition to reduced overall cell and vector requirements, illustrating that early application may resolve key obstacles to the widespread application of ATMPs for inherited disorders. Here, we provide a selective review of the latest ATMP developments for prenatal, perinatal and pediatric use, with special emphasis on its comparison with ATMPs for adults. Taken together, we provide a perspective on the enormous potential and key framework parameters of clinical prenatal and pediatric ATMP application.

1. General Introduction

Advanced therapies are based on innovative uses or the genetic manipulation of cell and tissue materials, and as treatments for human disease, are often without alternatives or are superior to treatments with conventional drugs. Be it for advanced or conventional treatments, due to regulatory, ethical and commercial pressures, numbers of medical products generally lag behind for pediatric compared with adult applications. However, in particular for advanced therapies, the case can be made that this “age gap” in drug development offers more harm than protection for patients, and that earlier-in-life application, from adult to pediatric or even to prenatal, would have tremendous medical, supply and commercial benefits. In this review, we present the corresponding background and arguments for pediatric and more recently conceived advanced prenatal therapies, by first outlining the current status and challenges of advanced therapies in general, before presenting the conceptual and regulatory framework for early interventions, followed by details for informative preclinical and clinical studies. After discussions of technical and non-technical elements and developments that might facilitate the further and more widespread success of early interventions, the article closes with corresponding perspectives and conclusions.

2. Current Status and Challenges of Advanced Therapy Medicinal Products (ATMPs) and Rationale for Early Interventions

2.1. Defining ATMPs

ATMPs are medicines that have begun to transform our ability to treat injury and disease. They are defined by the European Medicines Agency (EMA) as comprising gene therapy medicinal products (GTMPs), somatic cell therapy medicinal products (CTMP), tissue-engineered products (TEPs) and combined ATMPs as a combination of any of the three product categories [1]. Specifically for the EU, the EMA provides up-to-date information and relevant guidelines covering their classification [2] and toward marketing authorization [3,4], albeit with a functionally limited search interface. Likewise, clinical trials and new medicines are centrally registered in EMA databases [5,6], but are limited to products and trials approved through the centralized EU procedure, which can be bypassed by application to one or several nation states [7]. Differing terminology and definitions cover ATMPs and their subcategories outside the EU [7,8], which hinders systematic global assessments, but has not stopped an avalanche of comprehensive recent reviews covering their global development, application, regulation, risks and prospects [8,9,10,11,12,13,14,15,16,17,18,19]. For clarity, the present review will refer to advanced therapies in accordance with the most recent quarterly recommendations on the classification of ATMPs by the EMA [20]. Despite this European reference point, and although providing EU-specific references to regulatory procedures and public resources, we will cover findings and prospects for early interventions with a global perspective.

2.2. Current Key Areas of ATMP Application

In line with their wide-ranging definition, ATMPs may apply to a multitude of inherited and acquired diseases. However, although preclinical development for ATMPs has already touched on hundreds of conditions, only few major technologies and applications of ATMPs have progressed to the clinical trial stage or received marketing approval. Reasons for this can be found in inappropriate study designs (in terms of numbers, experimental groups, endpoints and comprehensiveness), limited transparency and comparability across studies, high cost and the slow political, regulatory and industry adoption of ATMPs [21]. Among a global pipeline of dozens of approved ATMPs and hundreds of ATMPs in development [22,23], the most advanced ATMPs with proven potential for early therapeutic intervention include receptor-engineered and chimeric antigen receptor (CAR) cells, ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs) [14]. Of these, CAR cells were initially conceived and developed over several generations of increasingly effective and durable CAR T cells [24], but are now also being developed as CAR natural killer (NK) cells [25] as powerful anti-cancer immunotherapeutic agents for autologous and allogeneic application, respectively [26]. HSPC-based ATMPs have enabled pioneering clinical autologous applications of gene therapies for both gene addition [16,27,28,29,30,31] and gene editing [32], whereas MSCs have also seen widespread use for allogeneic application in different tissues and in the modulation of immune responses (e.g., for Obnitix® and Alosifel®) [33,34]. Based on their diverse application and due to their exemplary level of development, as well as for prenatal and pediatric use, CAR cells, HSPCs and MSCs will thus serve as the main examples for ATMP progress and applications throughout this review. Importantly, many additional ATMPs may have great potential for early-in-life applications, but cannot be detailed here. For instance, the diverse category of TEPs, which are applied as autologous or allogeneic cell-based engineered tissues for the regeneration of skin, cartilage and bone, has great clinical significance [35]. However, its successful application in pediatric or prenatal settings is thus far limited to single case studies, safety assessments or small grafts; thus, systematic coverage of TEPs beyond selected landmark examples is outside the scope of this article.

2.3. Exemplary ATMP Successes

Among ATMPs under EU regulation, CAR-, HSPC- and MSC-based therapies may offer new prospects to improve the treatment of several conditions with unmet medical needs. For instance, CAR technology has been a trailblazer for processes and regulations governing personalized ATMPs and employs synthetic receptors to direct autologous immune cells to any cellular target without HLA restriction [36]. Current second-generation CAR T cells carry engineered receptors that encode the recognition domain for a tumor-specific epitope, bound via an optional scaffold domain to activation and costimulatory domains, which together facilitate target recognition, cytotoxicity and T cell expansion. The showcase application for CAR technology is autologous CAR T cells against the abundantly expressed and non-essential CD19 B cell marker which, after early success in clinical application to relapsed/refractory B cell malignancies [37], is the basis of currently approved CAR-based treatments (Tisagenlecleucel/Kymriah® EMA/FDA, axicabtagene ciloleucel/Yescarta® EMA/FDA, brexucabtagene autoleucel/Tecartus® EMA/FDA, lisocabtagene maraleucel/Breyanzif® EMA authorization pending/FDA) [24]. With ongoing attempts to target additional markers of malignancies and to even apply CAR technology to solid tumors, the most prevalent targets in addition to CD19 are the less abundantly expressed CD22 and CD20 for B cell malignancies [38,39] and the B cell maturation antigen (BCMA; by idecabtagene vicleucel/Abecma®) for multiple myeloma [40]. As additional developments for CAR T cells application, receptor components may be swapped in a modular fashion, site-specific delivery of CARs to the TRAC locus in T cells using clustered regularly interspaced short palindromic repeats (CRISPR) technology improves their potency and consistency of expression, and antigen escape by tumors may be reduced or the efficiency of therapy may be enhanced by the application of multiple CARs [41,42] or by bispecific CAR designs [43,44,45]. Finally, employing natural killer (NK) cells instead of T cells confers an improved safety profile by the avoidance of cytokine release syndrome and action on non-hematopoietic cells, and paves the way toward allogeneic (off-the-shelf) CAR cell applications because of the HLA-independent action of NK cells [46].
For HSPCs, autologous transplantation after genetic modification has emerged as the most popular and successful application of gene therapy as a viable option for a variety of monogenic disorders, possibly because of the long experience with allogeneic hematopoietic stem cell transplantation (HSCT) and the ability to manipulate those cells ex vivo with great efficiency [47]. For all these disorders, allogeneic HSCT remains the clinical standard for cure, but is limited by donor availability and associated severe immunologic complications (graft-versus-host disease (GvHD) and graft rejection) [48]. Genetically modified autologous HSPC products, mostly applied in the treatment of blood or immune system disorders, but also of several storage and metabolic disorders, represent an alternative and potentially safer one-off treatment option [49,50]. Despite continuous improvements in the manufacturing of modified cells (refined cell collection, isolation and culture protocols, better vector designs, safer tools based on gene/base editing), and an increasing number of products entering clinical trials for an expanding list of diseases, 30 years after the first ever gene therapy trial, only four HSPC-based GTMPs have been granted EU marketing approval: betibeglogene autotemcel (Zynteglo) for transfusion-dependent beta-thalassemia (TDBT); Strimvelis for severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID); atidarsagene autotemcel (Libmeldy) for metachromatic leukodystrophy (MLD); and elivaldogene autotemcel (Skysona) for cerebral adrenoleukodystrophy (CALD) [51,52,53,54]. All of the above therapies are based on ex vivo viral-vector-mediated gene transfer in HSPCs and the autologous transplantation of modified cells into patients. For in vivo applications of GTMPs, onasemnogene abeparvovec-xioi (Zolgensma) for spinal muscular atrophy (SMA), resamirigene bilparvovec (AT132) for X-linked myotubular myopathy (XLMTM) and voretigene neparvovec (Loxturna) for Leber congenital amaurosis (LCA), are EU-approved therapies based on the systemic or direct/topical delivery of viral vectors encoding functional copies of the respective disease-causing genes [55,56,57]. The above commercially available drugs are indicated either in pediatric-only or mixed pediatric/adult populations, because most of them concern early-onset severe or early-lethal inherited disorders.
Finally, MSCs have become the most clinically studied experimental cell therapy platform worldwide since their first evaluation in humans in 1995 [58]. Although initial insights into MSC properties and mechanisms of action were mainly gained from preclinical murine models and in vitro analyses of human MSCs, their application has already shown enormous potential in the moderation of immune and inflammatory reactions, bone diseases, cancer, and heart, liver or kidney failure, in part through paracrine and exosomal signaling. As exemplary fields of MSC application for immune and inflammatory reactions, acute GvHD (aGvHD) may lead to severe inflammatory reactions and the death of patients after allogeneic HSCT [59], and Crohn’s disease may lead to debilitating chronic inflammation of the gastrointestinal tract [34]. Likewise, acute or chronic injury, such as of the lungs, may irreversibly damage tissue by inappropriate immune responses and/or aberrant repair processes, usually leading to fibrosis and subsequent decline in organ function. MSC attenuation of inflammation prevents further injury and promotes repair [60,61,62], with frequent achievement of full aGvHD remission [63,64], improved bacterial clearance, potential differentiation of MSCs to replace damaged cells, and in part the cytokine-mediated, anti-inflammatory and pro-regenerative action of MSCs [65,66], all vindicating their therapeutic use. Regarding bone diseases, MSC-based therapies act by paracrine- and exosome-mediated signaling, in osteogenesis imperfecta in particular through action of exosomal RNA cargo [67,68], and in bone tissue engineering, scaffolds combined with osteogenically differentiated MSCs provide a better bone regeneration microenvironment and better bone growth than engineered cell-free scaffolds [69,70,71]. For cancer treatment, MSCs may show microenvironment-dependent pro- or anti-tumorigenic properties when unmodified, but clearly assume therapeutic properties when used for the exosomal delivery of cytotoxic agents after drug loading or after genetic manipulation [72,73]. In the treatment of heart, liver or kidney failure, MSCs variably mediate the stimulation of endothelial cells [74], inhibit apoptosis, inflammation and hepatic stellate cell activation [75,76], and deliver trophic factors and cell components by paracrine or exosome signaling and even cell fusion [77], respectively, in order to achieve therapeutic action.

2.4. Challenges of ATMP Application

Despite such prominent successes and progress, there are inherent limitations for the widespread use of ATMPs. Many factors determining the cost of ATMP development are those in common with the development of other drugs and treatments, whereas some key cost factors are unique to ATMPs. As for conventional drugs, observations for ATMPs and their efficacy under optimized laboratory conditions do not always reflect the complex and multifactorial reality in the clinic. Moreover, the complex nature of ATMPs and the direct link of their production with live cells from donors and/or recipients suggests that ATMP development is characterized by key challenges not encountered, or of different qualities from those faced, in the development of small-molecule drugs. The great diversity of ATMPs and their applications means that defining universal challenges is difficult; however, several key challenges applying to development, ethical and regulatory issues, supply and manufacturing, and marketing across many different ATMPs are outlined in Figure 1 and detailed in the current section.

2.4.1. Scientific and Medical

On the scientific or medical side, donor-specific features, incompleteness of our understanding of specific mechanisms of interactions with host tissues, and elusiveness of robust pharmacodynamic and pharmacokinetic models for different clinical applications represent major challenges that need to be overcome for each individual disorder to achieve successful clinical translation [49,78]. Especially for in vivo applications, the specificity and efficiency of target cell manipulation are problematic, whereas for ex vivo application based on HSCs, treatment-related morbidity related to myeloablation is a concern. For all ATMPs, addressing prenatal or early-onset diseases in a timely fashion and dealing with immune rejection of treatment and with pre-existing co-morbidities are common challenges.

2.4.2. Ethics and Regulatory

The translation of ATMPs from R&D to clinical trials and then commercialization faces unique ethical and regulatory challenges not encountered at all or to the same degree for small-molecule drugs. Ethically, the inherent complexity of ATMPs and the lack of precedents for many clinical studies are sources of uncertainty, and thus concern, for the safety of participants, whereas ethical considerations for trial participants also routinely prompt trial designs without control groups for what are often invasive procedures. Where inadvertent germline manipulation may be a concern in several in vivo applications, in particular of GTMPs, rare but high-profile illegal human germline manipulation has raised fundamental ethical concerns and have led to a high level of vigilance and stringent regulatory requirements, particularly for GTMP applications [79,80]. Moreover, regulatory requirements for ATMPs are generally high, and are moreover mutable for a nascent industry. Accordingly, ongoing evolution of regulatory, legal, quality-control and infrastructural aspects for ATMPs brings about that framework requirements across different legislatures differ greatly [8,81,82,83]; thus, regulatory challenges are seen as a key difficulty by manufacturers [84].

2.4.3. Supply

In addition to regulatory issues, access to bio- and good manufacturing practice (GMP) materials are a key impediment for ATMP applications. For cell-based therapies, suitable cells are not available in sufficient numbers for corresponding treatments, be it for autologous applications to a single patient or for allogeneic therapies, where the manufacturing of off-the-shelf ATMPs may aim to serve many patients. For certain disorders, the availability of suitable adult stem cells for autologous application presents a challenge even for abundant cells, such as HSPCs or MSPs, in particular where the disorders or injuries to be treated affect the abundance and viability of those stem cells, as is the case, e.g., for HSPCs in Fanconi’s anemia [16], where this may be addressed by improved mobilization and collection [85]. For allogeneic therapies, and even where culture protocols can be established that maintain the desired properties of the cells in question, bottlenecks may be addressed by advanced and scalable culture methods, although high single-dose requirements, e.g., for MSCs of up to 109 cells, or the rarity of starting material may continue to pose challenges for ATMP manufacturing for the foreseeable future [48]. The shortfall thus calls for improved identification, isolation, expansion and modification protocols for autologous application, and for scalable expansion technologies for off-the-shelf ATMPs [48], as is being argued and explored, e.g., for both HSPCs and MSCs in their countless applications [16,49,86,87]. Similarly, the pricing and availability of GMP reagents pose a problem for the clinical development of ATMPs, in that even well-funded clinical trials are held up by limiting global production capacities for GMP-grade reagents. The supply of GMP-grade materials was a limiting factor even before the general pharmaceutical supply chain issues brought about by the COVID-19 pandemic [88].
For many of the challenges of ATMP application, incremental improvements are being made; however, some key problems, particularly the limiting supply of GMP-level therapeutics at population scale, will require a landmark shift in the underlying technologies or in the way they are applied. Even a moderate increase in contemporary GMP requirements by expanding adult application of ATMPs would hardly be sustainable with current technologies [89].

2.4.4. Manufacturing

Supply shortages are aggravated by a lack of standardization of manufacturing and quality control procedures, which still pervades the nascent field of ATMPs, affecting the cost and predictability of production. More specifically for GTMPs, the large size and negative charge of nucleic acids and their consequentially poor penetration of cell membranes calls for the temporary removal of membrane integrity or for dedicated carriers to allow effective delivery to cells and tissues. The development of non-lethal systems for cell penetration and of effective vectors for transfer across membranes has been a long journey from initial attempts at plasmid transfection over advanced non-viral and viral delivery methods to a great diversity of permanent, transient and highly transient delivery of transgenes, of genome editing tools, and of RNA, nuclear reprogramming and epigenome editing reagents today, as we will point out for key developments later.
ATMPs based on autologous cell material are as variable as the patient population, are inherently personalized products that can only benefit from economies of scale in some aspects, and the place and time of manufacture have to be adjusted according to the patients. The corresponding personalized application of ATMPs also contributes to difficulties in trial design and preclinical evaluation as key scientific challenges. However, even for universal products and allogeneic application, variability, sensitivity, quality control and subsequent shelf life are issues of concern for ATMPs far more than for standard pharmaceuticals. Finally, the accessibility of therapeutic tools as targets for the patient’s immune system, and off-target delivery to cells and tissues, where those tools might be unproductive or even toxic, both represent a health and safety risk to patients, but also elevate GMP reagent requirements as an important bottleneck for the industry (see below). Altogether, manufacturing, quality standards and starting materials are therefore seen as key technical challenges for ATMPs [84].

2.4.5. Market and Pricing

On the financial side, funding and reimbursement are key challenges for ATMPs [84]. Be it the long development time combined with fast technology turnover, or be it the high inherent cost of manufacturing ATMPs combined with the uncertainty of manufacturing scale and reimbursement policies, the challenges for commercial ATMP development are enormous [12,90]. Most conventional drugs rely on the chronic application and correspondingly long-term reimbursement of manufacturers for development cost; however, most ATMPs are applied and paid as one-off treatments. The novelty of many ATMPs also means that both physicians and patients often need to be made aware of their existence and benefits, and that their integration into clinical routine, including post-treatment follow-up, often still needs to be established. Finally, just as regulatory requirements differ across legislatures, so do reimbursement models and agreements for ATMPs, which, in turn, creates uncertainty and causes hesitancy or absence of investment for production and commercialization. Therefore, the step from proof of principle to market is particularly difficult for ATMPs, and even for safe and efficient products reaching marketing authorization, the inability of patients or health systems to pay for advanced therapies might ultimately lead to commercial failure and market withdrawal [91].

2.5. The Rationale for Early Intervention

A multitude of shortcomings thus bring about that ATMPs currently cannot unfold their full potential while promising a new era of effective cancer treatments, potentially curative treatments for inherited diseases, and off-the-shelf regenerative medicines, among others. It turns out, however, that some of the key impediments to wider and effective ATMP application, including high reagent requirements, ineffective systemic delivery, poor accessibility of target tissues and pre-existing immunity, can be addressed by early intervention, i.e., by application at the prenatal stage, in infancy, in early to late childhood or in adolescence.
Many of the aforementioned challenges are brought about or exacerbated by the restriction of treatment to adults, which, for pioneering treatments and especially clinical trials, is a matter of course, unless the disease in question causes death or irreparable damage in early life stages. The resulting tradeoff for therapy development is that the underlying bioethical guidelines protect the unborn or young life, while at the same time often hiding the true potential of ATMPs by application in adults, where early-onset diseases may already have caused irreparable direct or pleiotropic damage, where affected tissues or therapeutically relevant cells are hard to access or rare, or where requirements for cell material (such as TEPs or stem cells) and GMP reagents are orders of magnitude above what may be required to achieve similar benefits in utero or in pediatric patients. A further argument for the prenatal or pediatric application of ATMPs is the innate proclivity for healing in younger patients and the generally superior regenerative performance of TEPs or stem cells earlier in life. Late treatments are thus limited in their effectiveness due to pre-existing damage and reduced graft performance, exacerbating the universal challenges of target-specific in vivo delivery and stem cell retrieval, and inflating the cost (see Section 6 and Section 7), which thus effectively limits the accessibility of ATMPs to those in need. In utero and pediatric therapies effectively improve all of these critical treatment parameters and may be instrumental in giving more patients and diseases the benefit of advanced therapies. Prenatal treatment, as compared with any postnatal treatment, brings various advantages, including a less developed and thus more tolerant and even tolerogenic immune system [92], higher accessibility of target organs, and reduced target tissue size and cell numbers, with correspondingly lower reagent requirements [93]. However, at a stage where early and in utero ATMP interventions, in particular, are themselves still in their infancy, researchers and clinicians are faced with a dilemma: the earlier the intervention, the greater the potential benefits, but also the greater the potential risks and uncertainties of ATMP applications (Figure 2).

3. Conceptual and Regulatory Framework for Early Interventions

Early interventions, although still universally under-researched compared with therapeutic applications in adults, may have an essential role to play in the enhancement of ATMP accessibility and effectiveness. Both pediatric and in utero treatments come with distinct drawbacks and benefits in their practical application, which are subject to resolution and enhancement by ongoing research and development efforts in the laboratory and clinic (see Section 4 and Section 5). Early interventions are correspondingly couched within a conceptual and regulatory framework that, in many aspects, differs quantitatively or even qualitatively from that applied for adult interventions. To avoid the erosion of ethical constraints for early intervention and to warrant benefits and prevent potential harm for patients, establishment, awareness and observance of that framework is essential.

3.1. Pediatric

In their pediatric application, ATMPs encounter the same routes of application and problems faced during adult application, but benefit from incremental efficacy, accessibility and cost benefits, and from decremental pre-existing infections and morbidities with lower age and body size, with additional pros and cons, as shown in Figure 2. On the regulatory side, pediatric medicines in the EU have assumed a special role since 2007, when the EU Paediatric Regulation came into force [94], comprising Regulations (EC) No 1901/2006 and its amendment No 1902/2006 [95,96] and aiming to achieve better product information and more pediatric medicines and pediatric research [6]. As a result, in its ten-year report on the implementation of the Paediatric Regulation, the EMA reported an increase in many therapeutic areas in pediatric medicines, albeit with little development for exclusively pediatric diseases or for diseases with distinct pediatric presentation [97]. Likewise, there was an only moderate increase in pediatric research in 2017, from 9.3% pediatric as a proportion of all trials in 2006, to 12.4% in 2016 [97,98]. Both the Paediatric Regulation and the ten-year report acknowledge the dilemma that protecting children from clinical trials for ethical reasons, and as a group that requires legal and regulatory protection, has resulted in a scarcity of specifically pediatric medicines, which has led to the widespread off-label use of adult medicines for pediatric patients, with inherent risks and uncertainties concerning optimal dosage, suitable modes of administration and age-specific side-effects. In recognition of this shortfall of pediatric treatments, the Paediatric Regulation therefore offers incentives for pediatric medicine development to compensate for the cost disadvantage associated with the requirement to investigate age-stratified action for inherently smaller patient populations than would be encountered for adult treatments. These incentives include six-month extensions on the supplementary protection certificate of medicines, and 12 instead of 10 years of market exclusivity for orphan medicines for compliant companies, supported by an advantageous pediatric-use marketing authorization (PUMA) and a specific pediatric expert committee and free advice to the industry from the EMA. This is paired with comprehensive measures and resources for the dissemination of pediatric studies, including an EU network of pediatric investigators, an inventory of pediatric needs, a public database of pediatric studies and the obligation for companies to submit any pediatric data for approved medicines to the regulators for analysis. In line with this and further promoting the dissemination of pediatric trial results, clinical trials for pediatric interventions in the EU are covered in their entirety from phase I to IV in the EU Clinical Trials Register, in contrast to adult interventions, for which phase I trials are excluded [6].

3.2. In Utero

In utero therapy offers the possibility of disease treatment and prevention before birth and is considered most appropriate after at least 7 weeks of gestation, when the primordial germ cells are relatively protected from inadvertent germline modifications [99,100]. It can be achieved either transplacentally, by introducing the medication into the maternal circulation, or by direct injection into fetal tissues or circulation. Both approaches call for procedures and reagents that are distinct from those applied in pediatric and adult patients. For transplacental in utero application, the placenta represents a key challenge, as both a route and an obstacle to transfer. A short-lived but constantly developing organ, the placenta separates the maternal and fetal circulations and, at the same time, enables communication between both entities. During approximately nine months of pregnancy, it serves many functions to support fetal development, including the transport of nutrients and gases, immune defense, endocrine signaling for hormone and transmitter homeostasis, and as barrier to protect the fetus against toxins from the maternal circulation [101]. Importantly, and while conventional pharmaceuticals of high liposolubility and low molecular weight traverse the placenta relatively easily, the placenta presents a substantial mechanical and functional barrier and possibly a toxicity target for therapeutics based on genes, cells, or tissue engineering. Therefore, alternatives to transplacental routes of administration are being explored, such as intravenous injection, intra-amniotic administration, or targeted drug delivery approaches [102]. One of the best-researched means of prenatal ATMP administration is ultrasound-assisted injection in the fetal umbilical vein, which allows injected gene constructs or stem cells to bypass the lungs, via the ductus arteriosus and foramen ovale, and to directly enter the fetal systemic circulation. Such fetal injection even offers significant advantages over the early postnatal intravenous administration of ATMPs into a peripheral vein, which often leads to the trapping of cells or gene carriers in the pulmonary microcirculation [103] and reduced systemic exposure. However, direct injection poses the challenge of accessibility and visualization of target tissues and of a developmentally changing anatomy, and the risk of treatment-related injury to mother and child, so that special surgical skills and equipment are required for the procedure [104,105,106]. Together with the unique safety and ethical considerations inherent to the in utero manipulation of human life, these technical requirements currently represent a substantial roadblock for the widespread application of in utero therapies, with additional pros and cons, as shown in Figure 2. On the regulatory side, EU documents currently refer to ATMPs in connection with in utero transfer mostly in the context of inadvertent germline transmission [107]. Importantly, however, the recent EC Guidelines on Good Clinical Practice specific to ATMPs explicitly acknowledge the possible necessity of in utero intervention in severe early-onset conditions or where only early treatment offers benefit, and in general terms recommend the adoption of additional safeguards appropriate for the product, disease and developmental stage [108]. Meanwhile in utero treatments are already applied using conventional drugs, such as for fetal arrhythmias by digoxin or sotalol [109] or for the prevention of fetal viral infection by antiretrovirals in HIV-positive women [110]. Based on growing experience for in utero non-ATMP treatments and from preclinical ATMP studies (see Section 4), it can therefore be hoped that the clinical translation of in utero ATMP applications is not too far in the future [93].

4. Preclinical Studies of Early Interventions in Animal Models Using ATMPs

4.1. Pediatric

The pharmacological profile of a new medicinal product is generally assessed in adults prior to testing in the pediatric patient population, and efficacy and safety data are extrapolated to children [111]. However, substantial differences in the disease pathology and pharmacological properties of many ATMPs often exist between adults and children, thus necessitating studies in juvenile animal models prior to pediatric application. Alas, dedicated pediatric studies in animal models are few and far between, also because the juvenile time window for mice (the most versatile and most widely used model organism) is fairly narrow, with mice reaching sexual maturity from as early as 23 days after birth for females and usually from 6 weeks for males [112]. As highlighted in a Special Issue assessing pigs as model animals [113], corresponding research therefore greatly benefits from alternative animal models with an extended juvenile period, such as dogs, pigs, sheep and non-human primates [105,113,114,115,116,117,118], which may moreover serve as a large animal model for in utero applications. Importantly, many murine studies of early-onset diseases, although stretching into adult application, are representative of early interventions and therefore also informative in the present context.
Outside our focus on CAR-, HSPC- and MSC-based ATMPs, early treatments in a cornucopia of rodent and large animal models have already been highly informative for a range of ATMPs, target tissues, diseases and juvenile stages, as may be exemplified here, with treatments based on adeno-associated-virus (AAV)-mediated in vivo gene addition. Recent examples include applications in 2–4-month-old sheep to treat Tay–Sachs disease [118] and in early postnatal and juvenile mice to treat CLN3 Batten disease [119], as two exemplary lysosomal storage disorder, as well as in 12-week-old dogs to treat X-linked retinitis pigmentosa [120], and in early postnatal mice, which mimic the human fetal inner ear, to treat congenital hearing loss and vestibular dysfunction in over a dozen studies [121].
For CAR cell applications, the predominance of CAR-T cells and their autologous application largely shifts analyses to toxicity-only assessments in pure animal models, or to more comprehensive functional assessments in murine xenograft models. For the latter, the age of human cell donors is usually not disclosed and recipient NSG mice are usually adult; thus, corresponding publications to date do not allow conclusions drawn from comparisons of the relative performance of early life and adult interventions. Importantly, the contemporary predominant application of CAR cells is cancer treatment by autologous CAR T cells, the performance and developmental age of which are bound up with the age and state of the affected patient. However, in particular for the nascent allogenic application of, e.g., CAR-NK cells, comparative analyses for performance of adult vs. juvenile cells in xenograft models would be extremely informative, because the emergence of standardized protocols and impressive efficacy data indicate for the latest CAR-treatment-based strategies [122]. For instance, the evaluation of second- and third-generation anti-CAIX CAR-T cells in NSG-SGM3 mice transplanted with CAIX-expressing clear-cell renal cell carcinoma skrc-59 cells achieved complete remission and tumor-free survival on day 72 after treatment for the best combination of CD4/CD8 CAR-T cell ratio and CAR-T receptor [123]. Evaluation of cord-blood-derived fourth-generation anti-CD19 CAR-NK cells engineered for enhanced cytokine signal transduction achieved the virtually complete suppression of tumor growth in Raji (Burkitt-lymphoma-cell)-transplanted NSG mice, allowing up to 341 days of tumor-free survival, against the death of all control animals in under 1 month [124]. Finally, in the first application of Vδ1 γδ CAR-T cells, targeting GPC-3 as a frequent and abundant marker of solid tumors slowed tumor growth after transplantation of HepG2 cells into NSG mice, down to below 10% of growth in controls, for a test period of over 30 days [125]. This was achieved in the absence of GvHD and toxicity symptoms, because γδ CAR-T, similarly CAR-NK cells, acts HLA-independently and could thus be used as off-the-shelf ATMP.
For HSPCs, murine xenograft models have been crucial for the development and clinical translation of autologous and allogeneic HSPC-based therapies, including gene therapy, since the late 1990s [126]. Immunocompromised mice, humanized mice and genetically engineered disease mouse models have been used to address key bottlenecks of ex vivo and in vivo application of HSPC-based ATPMs, including the maintenance of HSPC multilineage potential, efficient engraftment of cells and safety [127]. Neonatal and juvenile mouse models, in particular, have been extremely useful in the development of HSPC-based gene therapy for primary immunodeficiencies, where early intervention is a sine qua non. For this, the adenosine deaminase deficiency (ADA)-/- neonatal mouse model was used in preclinical studies to assess efficiency and safety of ADA-carrying lentiviral vectors by ex vivo transduction and the autologous transplantation of modified HSPCs, efforts that ultimately led to the EU marketing authorization of Strimvelis in 2016 [128,129,130]. HSPC-based ATMPs have also been tested in neonatal mouse models of further disorders, such as Wiskott–Aldrich syndrome, mucopolysaccharidoses and other storage disorders [131,132,133]. An exceptional achievement is the humanized transgenic thalassemia mouse models developed by the Ryan group which, in contrast, to knockout models, develop transfusion-dependent thalassemia major, and thus are the most faithful representation of the disease and of therapeutic efficiencies in vivo, from in utero to adult applications [134,135,136,137,138]. Larger animal models have also been applied in cases where mouse knockout models have failed to faithfully recapitulate the disease phenotype or where longer-term follow-up has been essential [139]. Among others, juvenile canine leukocyte adhesion deficiency and X-linked severe combined immunodeficiency (X-SCID) dog models, as well as juvenile nonhuman primate X-SCID and human immunodeficiency virus (HIV) models, have been crucial in advancing HSPC gene therapy based on both gene-transferring viral vectors and newer gene editing tools [114,117].
For MSCs, many preclinical trials have been completed and many more are currently ongoing to explore their safety and efficacy in a wide range of acute and chronic disease models, complemented by studies merely investigating the (non-ATMP) application of MSC-derived exosomes and extracellular vesicles. A fundamental limitation for purely murine models (as opposed to xenograft models) in ATMP research is the profound differences for key aspects of MSC biology in ATMP development between murine and human MSCs and the correspondingly poor representation of human therapy applications in studies based on mouse MSCs [140]. These differences concern cell expansion behavior, properties after immortalization or cryopreservation, and choices of paracrine signaling molecules [140,141]; moreover, conclusions across multiple studies are exacerbated by differences between alternative models for the same disease [142]. Therefore, with the swift elimination of human MSCs in immunocompetent mice, xenograft studies based on immunodeficient mice or studies in species for which MSCs or the relevant anatomy more closely reflect human biology [143,144] appear to be the most informative. Based on adolescent to young adult (6–8-week-old) mice, the latest findings for immune and inflammatory disorders include the identification of therapeutic antioxidant and pro-angiogenic action of placenta-derived human MSCs in a surgical model for Crohn’s-like enterocutaneous fistula [145]. Another recent study demonstrated enhanced colonic homing and the enhanced induction of macrophage IL-10 release, through transient CXCR2-receptor and semaphorin-7A expression on human MSCs, respectively, in an immunocompetent murine chemically induced inflammatory bowel disease model [146]. For recent MSC use in GvHD, culture-expanded, high-dose human umbilical-cord-blood-derived (UCB) MSCs were simultaneously transplanted with same-donor UCB HSPCs into NSG mice to effectively suppress GvHD and achieve 60 days of event-free survival [147]. As a particularly striking endorsement of MSCs as therapeutic agents for injury and inflammation, a recent systematic review on preclinical studies testing cell-based therapies in experimental neonatal lung injury, mainly applied in a hyperoxic rodent model of bronchopulmonary dysplasia (BPD), identified MSCs from among 15 distinct cell-derived therapies as the most effective cell-based therapy for key outcomes [148]. BPD in hyperoxia models has been studied for therapies based on MSCs [149]. When delivered intravenously, intraperitoneally or intratracheally, MSCs attenuated neonatal lung injury by decreasing lung inflammatory mediators, such as IL-6 and TNF-a, and reducing the expression of angiotensin II, angiotensin II type 1 receptor, and angiotensin-converting enzyme [149]. MSCs also improved alveolar structure and angiogenesis, inhibited lung fibrosis, and improved exercise capacity in animal models of BPD [150,151,152,153]. Similarly to BPD, acute respiratory distress syndrome (ARDS), which represents a global medical concern with significant morbidity, might also be ameliorated by MSC-based therapies [154]. Experimental in vivo models of lung injury, including acute lung injury (ALI) and ARDS, demonstrated the therapeutic efficacy of MSCs [155,156] or their exosomes, vindicating the corresponding application of MSCs in clinical trials.
Finally, and across different cell and vector systems, several studies in animal models have addressed age comparisons for the efficiency of candidate ATMPs [157,158,159,160,161,162]. For instance, direct AAV-mediated delivery of the CLN2 gene for treatment of the lysosomal storage disorder, late infantile neuronal ceroid lipofuscinosis, showed clear efficiency advantages for pre-symptomatic compared with post-symptomatic application [160], and a clear survival advantage for younger recipients, with effectively doubled survival time from 2-day- to 3-week- and 7-week-old recipients [157]. For treatment of spinal muscular atrophy, intravenous injection of an SMN-encoding AAV in neonate (postnatal day 1) diseased mice rescued neuromuscular phenotype and life span, in contrast to treatment of 10-day-old mice, followed up by corresponding injection and motoneuron transduction in a neonate wildtype cynomolgus macaque [159]. For leukodystrophy Canavan disease, intravenous AAV injection to deliver the AspA gene on postnatal days 0, 6, 13 and 20 retained significant therapeutic action up to day 20, but for all parameters tested gave increasingly better restoration of normal performance with decreasing age at treatment [158]. Finally, young adult vs. aged rats were recently transplanted with bare and allogeneic-MSC-coated vascular grafts to demonstrate superior performance (e.g., for graft integration, blood flow, neotissue density, collagen fiber density and orientation) in young vs. aged recipients and for MSC-coated vs. bare grafts [162], which gives clear implications for MSC inclusion and, despite absence of juvenile animals in the study, once again for a general positive effect of application in younger recipients.

4.2. In Utero

Recent years have seen significant progress in the in utero application of different classes of ATMPs. Transfer of stem cells is rarely used, whereas in utero ATMP application by the AAV-mediated direct delivery of gene editing components or therapeutic transgenes is of particular prominence, although direct injection into fetal yolk sac vessels [163,164], ionizable lipid nanoparticles (LNPs) [165] and even in utero electroporation [166] have also been employed to achieve the delivery of cargoes such as mRNA, editors or gene addition components.
Exemplary studies for direct AAV-mediated delivery include early achievements of sustained reporter gene expression in the pulmonary epithelium after injection in the amniotic sac [167] and tolerance induction by the delivery of human factor IX in hemophilia B mice [168]. AAV was also used for the delivery of adenine base editors for efficient correction in the liver and heart and low-level correction in the brain in Idua(W392X) mutant (Hurler syndrome) mice [169] and that of a β-glucosylceramidase transgene by fetal intracranial injection in Gba knockout (Gaucher disease) mice [170] for lysosomal storage disorders. Similarly, CRISPR/Cas9- and cytosine base editors were delivered by intravenous in utero injection to establish proof of principle for in utero editing to modify proprotein convertase subtilisin/kexin type 9 (PCSK9) as a target for coronary heart disease and to correct 4-hydroxyphenylpyruvate dioxygenase (Hbd) as therapy for hereditary tyrosinemia type 1 [171]. AAV8-mediated delivery of human factor IX enabled the long-term correction of hemophilia in cynomolgus macaques [105], as did the AAV5- and AAV8-mediated delivery of human factors IV and X [172] in both studies, largely due to randomly integrated provirus in hepatocytes. Finally, AAV2-mediated delivery of MSRB3 by transuterine microinjection into the otic vesicle of MsrB3 knockout embryos has been used to address hearing loss and vestibular dysfunction, which has also been addressed, e.g., by plasmid-based delivery combined with electroporation in connexin 30 knockout embryos [121]. Non-AAV-based direct injection of therapeutic agents has additionally been applied using an adenoviral vector to deliver CRISPR/Cas components and inactivate a mutant SftpcI73T gene for partial disease correction in a mouse model of monogenic lung disease [173]. Direct in vivo vector delivery was also performed by the intrahepatic fetal injection of HBB-encoding GLOBE lentiviral vector to achieve the correction of β-thalassemia in a humanized mouse model [137], for comparison with the intraperitoneal delivery of wild-type HSPCs with overall low correction efficiencies [174], and by the intra-amniotic injection of polymeric nanoparticles loaded with triplex-forming peptide nucleic acids and single-stranded donor DNA as gene editing components in HBBIVS2−654 thalassemic mice [175].
Concerning ATMP development based on in utero stem cell transplantation, ex vivo gene addition of human factor VIII to placenta-derived MSCs allowed the detection of postnatal transgene expression after in utero transplantation into wild-type mouse embryos, as proof of principle for a potential corresponding hemophilia A therapy [176]. For HSPCs, in utero application would be based on long-established in utero hematopoietic stem cell transplantation [177] which, due to immunological immaturity [92], can even be employed as conventional fully allogeneic HSCT [177], but in the absence of in utero conditioning regimens, only achieves the mixed chimerism of donor cells for allogeneic or autologous application [177,178]. In this context, amniotic-fluid-derived stem cells show superior performance as a potential substrate for future in utero therapies [179,180]

5. Clinical Studies of Early Interventions

5.1. Pediatric

A large number of pediatric clinical trials are in progress for ATMPs, and in some cases have already led to approved treatments (Section 2). For TEPs, several studies and applications of tissue engineering for the treatment of skin and soft tissue damage provide notable landmarks with great potential for pediatric application [181,182,183], as was recently demonstrated in clinical studies, e.g., for epidermal autografts (JACE) [184] and composite skin allografts (Apligraf) [183,185]. In the context of genetic skin disorders, two pioneering pediatric studies in seven-year-old boys with junctional epidermolysis bullosa combining retroviral gene addition in autologous keratinocyte stem cells with tissue engineering demonstrated the complete functional regeneration of limited epidermal grafts [186] and permanent clonal reconstitution of the entire epidermis, respectively [187]. These studies at the University of Modena and Reggio Emilia have provided the first concept and then mechanistic insights for wider clinical evaluations of genetically corrected autologous epidermal grafts to treat other genetic disorders, such as Netherton syndrome [188] and recessive dystrophic epidermolysis bullosa [189]. Noteworthy outside our focus on CAR-, HSPC- and MSC-based therapies are also ATMPs with pediatric application developed under the EMA Priority Medicines (PRIME) scheme [190]. Corresponding pediatric treatments still under investigation and pending approval include rebisulfigene etisparvovec (ABO-102), an AAV9-based gene therapy drug for the in vivo treatment of mucopolysaccharidosis IIA-Sanfilippo syndrome, and beremagene geperpavec (KB103), an HSV-1 collagen-expressing vector for the topical treatment of dystrophic epidermolysis bullosa, both of which are currently in clinical trials (NCT04088734, NCT04360265, NCT02716246 and NCT03536143, NCT04491604, respectively). A third pediatric ATMP, AT-GTX-501, based on an AAV9 vector containing the human CLN6 gene to slow disease progression in variant late infantile neuronal ceroid lipofuscinosis 6, has recently been discontinued because it failed to stabilize disease progression in long-term follow-up (NCT04273243 and NCT02725580) [191].

5.1.1. CAR Cells

For CAR cell application, CAR-T cell-based therapy is exceptionally advanced and too prolific to cover here in detail for all corresponding clinical trials, with marketing approval for several ATMPs for pediatric application (see also Section 2). Notable here is the EMA and FDA ACCELERATE collaboration for pediatric cancer patients [192] which, among other ATMPs, led to marketing approval as priority medicines under EMA’s PRIME scheme [190] for the CD19-targeting, CAR-T-based tisagenlecleucel (Kymriah®, e.g., NCT02529813 and many others) for pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia, whereas for other equivalent treatments, such as axicabtagene ciloleucel (Yescarta®, e.g., NCT02348216 and many others), data are still pending that would warrant treatment in pediatric patients. Overall, over 60 CAR-T trials for pediatric application are currently open, recruiting or ongoing (see, accessed 21 February 2022), with targets such as acute lymphoblastic leukemia (anti-CD19, anti-CD22, anti-CD19/22, anti-CD20/19), CNS tumors and sarcomas (anti-B7H3, anti-HER2, anti-EGFR806), Hodgkin lymphoma (anti-CD30), liver cancer (anti-GAP), neuroblastoma (anti-GD2), myeloid leukemia (anti-CD33, anti-CD123) and T cell lymphoblastic leukemia (anti-CD7).

5.1.2. HSPCs

For advanced HSPC-based therapies, promising preclinical data have led to dozens of products entering clinical trials during the last few years. Specifically, for early-lethal inherited disorders, clinical trials of related drugs exclusively involve children; otherwise, a mixed adult/pediatric or, more often, an adult-only cohort is used. The juvenile application of HSPC-based ATMPs in children, mostly as gene therapy of inherited disorders, comes with its unique challenges and opportunities [193]. Early application is paramount for many HSPC-based GTMPs, especially when the corresponding disease poses an immediate threat to life or causes early irreversible damage, but also because early drug administration is associated with better outcomes due to better health status, bone marrow (BM) condition and quality of stem cells [194,195]. This benefit of earlier intervention, highlighted in a plethora of preclinical studies in animal models (see Section 4), as well as in clinical studies of allogeneic HSCT [196], has only recently begun to show in gene therapy clinical studies, strongly advocating for improvements in prenatal screening and early diagnosis [197]. Moreover, early treatment reaches therapeutic effects at a lower price (lower body mass requiring lower drug dose), abolishes or reduces long-term medication requirements and prevents disease complications, together making an otherwise expensive and inaccessible treatment rather cost effective [198,199,200].
Two out of the three EU-approved HSPC-based GTMPs, Strimvelis for ADA-SCID and atidarsagene autotemcel (Libmeldy) for MLD, are specifically indicated in young pediatric patients [201,202], whereas betibeglogene autotemcel (Zynteglo) for TDBT is indicated in children >12 years old and adult patients [197]. Lybmeldy in particular was given authorization for use only in children with late infantile or early juvenile MLD who are asymptomatic or have initial symptoms but can still walk independently and do not show mental deterioration, as the drug showed much less benefit in children with a more advanced disease stage [54,98,202]. As yet another advocate for early intervention, a recent gene therapy study for β-thalassemia, employing autologous HSPCs after lentiviral vector-mediated HBB gene addition in patients, including pediatric patients, allowed the direct comparison of pediatric and adult treatment with HSPCs and showed that a younger age is associated with better clinical outcomes (NCT02453477). This was attributed to an impaired BM microenvironment (as the recipient tissue of modified stem cells) and HSPC repopulating capacity in older patients, due to both aging and an advanced disease pathology [194,203].
Due to the large number of clinical studies involving HSPCs as GTMPs, Table 1, in addition to selected studies employing MSC, lists only studies involving products with pediatric application that are currently under the EMA PRIME scheme.

5.1.3. MSCs

The success of MSCs in preclinical models has, over the past 10 years, prompted investigation of their regenerative potential for stem-cell-based therapies in the treatment or prevention of GvHD and in different lung diseases of infants and children, such as BPD, pneumonia, ALI and ARDS [204].
GvHD. Typical for the pediatric development of new medicines, many products successfully applied in adult patients do not or only with delay find application in pediatric patients. For instance, whereas for Alosifel® (aka Darvadstrocel) no data exist for patient groups from 0 to 17 years [205], Obnitix® has been employed successfully in pediatric patients with steroid-refractory acute graft-vs-host disease (aGvHD) [33]. Despite this relative shortfall of pediatric development, a gratifyingly large selection of clinical studies has nevertheless already shown the benefit of allogeneic MSC treatments for aGvHD correction or prevention in pediatric patients or for the promotion of engraftment [63,206,207,208,209,210,211]. Of note, the source and nature of the MSCs applied affected the outcome, in that the co-infusion of MSCs effectively prevented aGvHD, but umbilical-cord-blood-derived MSCs additionally improved engraftment, in contrast to parental-derived MSCs [212,213].
BPD. BPD is a main complication of prematurity, resulting in significant morbidity, mortality and lifelong consequence of early impairment [214], emphasizing the potentially critical role of early intervention. An apparent reduction in the BPD of lung-resident stem/progenitor cells from the endothelial, mesenchymal and epithelial lineages [215,216] as possible cause for lung growth has suggested a potential therapeutic role of MSCs in preterm infants. This prompted a pioneering phase I dose-escalation trial based on treatment with umbilical-cord-blood-derived MSCs in nine preterm infants at high risk of BPD, which was well tolerated without any serious adverse effect (NCT01297205) [217]. A subsequent two-year follow-up study confirmed the lack of any long-term side effect in the treated patients [218], which has now been extended up to 5 years of age (NCT02023788) [219]. A similar phase I dose-escalation trial on the safety and feasibility of intratracheal MSCs in twelve preterm infants at high risk of BPD yielded comparable results (NCT02381366) [220] as encouragement for further trials including controls and a greater sample size.
ARDS. ARDS is a life-threatening condition with acute hypoxemic respiratory failure and other cardio-pulmonary features [221], with a range of possible environmental causes and underlying conditions [222] and the contribution of dysregulated inflammatory/immune responses, coagulation, and alveolar membrane permeability in its pathogenesis [223]. Pediatric mortality is high [154] and supportive care is inadequate, which renders ARDS an ideal target for evaluations of pediatric MSC treatment. Three adult-only trials (phases 1, 2A and 2B) have given encouraging results for an intravenous dose of 106 cells/kg (NCT01775774, NCT02097641 and NCT03818854) [224,225,226], with the need to improve MSC viability and to demonstrate safety and efficacy in pediatric patients.
Table 1. Exemplary clinical in utero and pediatric trials of ATMPs.
Table 1. Exemplary clinical in utero and pediatric trials of ATMPs.
Cell TypeTarget DiseaseDrug 1Drug Short DescriptionNCT IDIU/P/AnRef.
CAR-T cellRelapsed/Refractory HLCD30.CAR-TCD30-directed genetically modified autologous T cellsNCT04268706P/A14 (recruiting) [227,228]
HSPCTDBTCTX001Autologous CRISPR-Cas9 modified ex vivo CD34+ cellsNCT03655678P/A15[229,230]
HSPCSCDCTX001Autologous CRISPR-Cas9 modified ex vivo CD34+ cellsNCT03745287P/A7[229,230]
HSPCTDBTOTL-300Autologous CD34+ cells transduced ex vivo with a lentiviral vector (GLOBE) encoding the HBB gene.NCT02453477
HSPCLAD-1RPL-201Autologous CD34+ cells transduced ex vivo with a lentiviral vector (Chim-CD18-WPRE)encoding the ITGB2 gene NCT03812263P/A7[233,234,235]
HSPCMPS-IHOTL-203Autologous CD34+ cells transduced ex vivo with a lentiviral vector (IDUA LV) encoding the IDUA gene.NCT03488394P8[236,237]
HSPCXSCIDMB-107Autologous CD34+ cells transduced ex vivo with a lentiviral vector (CL20-i4-EF1α-hγc-OPT) encoding the IL2RG gene.NCT03315078
5 (recruiting)
8 (recruiting)
HSPCSCDECT-001-CBUM171-expanded cord bloodNCT04594031P/ARecruiting[242]
HSPCHigh-Risk Myeloid MalignanciesECT-001-CBUM171-expanded cord bloodNCT04990323P/ARecruiting[243]
HSPCFARP-L102Autologous CD34+ cells transduced ex vivo with a lentiviral vector (PGK-FANCA-WPRE) encoding the FANCA gene.NCT03814408
25 (recruiting)
5 (recruiting)
T cellSerious viral infections in allogeneic HSCT recipientsPosoleucel (ALVR-105)Allogeneic multi-virus specific T lymphocytes NCT04693637
12 (recruiting)
MSCBPDPneumostem®Intratracheal delivery of umbilical cord MSCs, 1–2 × 107 cells/kg BWNCT01297205
33 (T) + 33 (C)
MSCMMCPMSC-ECMPlacental delivery of PMSC-ECMNCT04652908
IU35 (T) + 20 (C) (recruiting)[255]
MSCOIBoost cellsIntravenous injection of first-trimester-derived allogeneic expanded fetal liver MSCsNCT03706482
IU/P15 (T) + 15 (C)
1 Treatments with in utero or pediatric aspects under clinical investigation and currently supported by the EMA Priority Medicines scheme (access date, 1 February 2022), with the exception of MSC-based studies. A: adult; BPD: bronchopulmonary dysplasia; BW: body weight; C: control arm; CAR: chimeric antigen receptor; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-Cas9; FA: Fanconi anemia; HL: Hodgkin lymphoma; HSCT: hematopoietic stem cell transplantation; HSPC: hematopoietic stem and progenitor cell; IU: in utero; LAD-1: leukocyte adhesion deficiency type 1; MMC: myelomeningocele (spina bifida); MPS-IH: mucopolysaccharidosis type IH (Hurler syndrome); MSC: mesenchymal stromal cell; n: number of participants (+ control/reference-treated patients); OI: osteogenesis imperfecta (brittle bone disease); P: pediatric; PMSC-ECM: placental MSCs seeded on an extracellular matrix; SCD: sickle cell disease; T: treated arm; TDBT: transfusion-dependent beta-thalassemia; XSCID: X-linked severe combined immunodeficiency.

5.2. In Utero

Due to uncertainties and bioethical concerns associated with nascent technologies of in utero ATMP application, few prenatal therapies are currently in clinical trials, and those that are aim squarely at preventing or ameliorating severe diseases with in utero onset, rather than reducing costs or increasing therapeutic efficiency in diseases otherwise suitable for postnatal ATMP application. In addition to animal studies for in utero ATMP application (see Section 4), non-ATMP in utero therapies such as curative intra-amniotic administration of ectodysplasin A protein for hypohidrotic ectodermal dysplasia [257] have paved the way for registration of the first in utero ATMP clinical trials.
For HSPC application, hydrops fetalis in alpha-thalassemia major represents a textbook case of the need for and unique potential of in utero therapeutic applications. With in utero blood transfusions as a powerful life-saving technology [258], in utero HSPC transplantation was a logical next step in a conventional HSCT trial with planned enrolment of 10 patients (NCT02986698) [259,260], and possibly paving the way for pioneering in utero gene therapy applications in the clinic, based on autologous HSPCs.
Drawing on MSCs, the in utero transplantation of placental MSC is envisaged in a trial addressing myelomeningocele (aka spina bifida; NCT04652908) as supportive treatment for successful outcomes of in utero myelomeningocele surgery, with the planned enrolment of 35 patients for combined treatment and 20 patients as surgery-only controls [255]. Based on allogeneic fetal expanded MSCs, another study addresses osteogenesis imperfecta (NCT03706482) with administration of three doses in utero in the treatment group vs. three doses starting at 4 months after birth for the control group [256].

6. Tools for Success of Early Interventions

In addition to benefits concerning therapeutic efficacy specifically for ATMPs, juvenile treatments in general cater for a large and growing market, as detailed elsewhere in this Special Issue [261], and as for any research and medical sector, growing application will lead to the creation of additional resources. In this respect, progress across all aspects of ATMP development will benefit from early interventions, but of particular importance here might be the recent developments for cell sources, vector development, and in particular, the burgeoning field of nanomedicine, as detailed subsequently.

6.1. Sources for Cell-Based Therapies

For CAR cells, T and NK cells as the substrate for the generation of CAR-T and CAR-NK cells, respectively, have a variety of abundant sources. For T cells, typically autologous peripheral blood mononuclear cells (PBMCs) are collected by leukapheresis before the isolation of T cells by CD3 selection [262]. T cells then require activation before transduction to create CAR-T cells, and activation is most reproducibly achieved with anti-CD3/anti-CD28 antibody-coated beads. Transduction is then typically performed by lentiviral or γ-retroviral vectors, with the preference of lentiviral vectors due to their safer integration profile [263] and effective transduction of quiescent cells [264]. For NK cells with their HLA-independent action, autologous but also allogeneic cell sources are suitable, including induced pluripotent stem cells, NK cell lines, umbilical cord blood or allogeneic PBMCs [265]. Depending on the cell source, such as PBMCs, irradiation of CAR-NK cells may not be necessary, whereas for other cell sources, such as the commonly used, cytotoxic and highly passaged NK92 cell line, irradiation is required to prevent malignancies [266]. Based on PBMCs, CD3-negative followed by CD56-positive selection is usually employed to isolate NK cells before activation and transduction. Importantly, off-the-shelf NK cell lines for specific malignancies are already available, such as CD38/BCMA-targeting FT576 line for multiple myeloma [267], and the CD19-targeting FT596 line for B cell malignancies [268].
In HSPC-based cellular therapies, BM, mobilized peripheral blood and umbilical cord blood are all popular sources of HSPCs, each with their own set of advantages and disadvantages, determined by differences in collection procedures, cellular content (cell types and numbers) and outcomes of transplantation [269]. The same tissues serve as HSPC sources for ATMP-related pediatric applications; however, children as donors or recipients of HSPCs face their own unique challenges [270,271,272]. For decades, BM has been the gold standard source of HSPCs in children; however, in recent years, an increasing number of centers has instead used mobilized peripheral blood as the primary source of HSPCs [273]. The procedure is less invasive than BM harvesting, and also characterized by the more rapid engraftment of HSPCs after transplantation [274]. For the collection of mobilized peripheral blood, mobilization agents such as G-CSF and plerixafor are administered to the patient to allow the rapid egress of HSPCs from BM into peripheral blood, from where they are then collected by a blood cell separator (apheresis machine) [275]. The procedure is more challenging in children, especially in those under 10 kg (higher risk of hypovolemic shock, hypocalcemia, hypervolemic cardiac overload and adverse events related to insertion of dialysis catheters), requiring more time and money than in adults [272]. There is, therefore, considerable scope for adjustment to pediatric needs, both for current procedure protocols and for existing adult-oriented technology, such as standard apheresis machines with their large extracorporeal volume in relation to the total blood volume of small children [276]. Even though higher HSPC yields are obtained during harvest in children compared with adults, the scarcity of stem cells with long-term repopulating potential (present among large numbers of committed progenitors or mature blood cells) in the available sources requires the enrichment and expansion of those cells in cultures to achieve therapeutic doses of products. Improvement and innovation in every step of source cell processing, from mobilization (newer mobilizing regimens to maximize HSPC harvest) to apheresis (to increase cell yields and minimize associated risks) and cell culture procedures (to retain stemness and long-term repopulating capacity of cells) are needed for the industrial translation of HSPC-based therapies [277,278,279].
MSCs are a heterogeneous subset of multipotent adult stem cells present in multiple tissues of different sources. Human MSCs can easily be isolated from the umbilical cord, BM, and adipose tissue, and when expanded in vitro can differentiate into different mesodermal cell linages with exceptional genomic stability and few ethical issues [280]. These characteristics have marked their importance in cell therapy, regenerative medicine and tissue repair. BM and adipose tissue are well characterized and documented sources of MSCs. When selecting adequate sources, clinicians should consider some practical limitations concerning the difficulty and invasiveness of the procurement process and various donor characteristics [281]. For BM-derived MSCs, harvesting these cells is a painful, invasive procedure, with a risk of viral exposure and with a potential reduction with donor age in the number, differentiation potential and maximal life span of BM-derived MSCs [282]. Instead, a large number of MSCs can be obtained from the adipose tissue through minimally invasive lipoaspiration methods [283], which maintain their potency with increasing donor age and possess a more robust immunomodulatory capability than BM-derived MSCs [284]. Finally, umbilical cord (UC)-derived MSCs exert faster self-renewal properties than BM-derived MSCs and can be obtained with a painless collection procedure from Wharton jelly, veins, arteries, the umbilical cord lining and the subamnion and perivascular regions [285].
Of note, for off-the-shelf application of any cell type, cell material needs to be expanded, which may be enhanced by new developments in advanced expansion technologies. This might also be necessary for autologous applications, where the underlying disease condition affects cell yield and function. A key step here is a transition from yield-limiting planar to multi-layer and target-cell-optimized microcarrier systems, where porous microcarriers may be of particular benefit for MSC expansion [48]. Alternatively, early ATMP application, with its ballpark drop in reagent requirements and cost, may effectively address the challenge of providing sufficient cell material instead.
Once collected and possibly expanded, cells of interest need to reach their application target, a process for which the selection of preclinical and clinical studies in Section 4 and Section 5 indicates a range of different successful cell delivery modalities for early interventions. Accordingly, intravenous injection readily allows CAR-T and -NK cells to find their engineered, receptor-specific targets, and allows HSPCs, MSCs and many other cell types to home to their tissue of origin. Alternatively, the intraosseous application of HSPCs has advantages for the speed of reconstitution [194], and targeting of cells to the CNS (where generally direct vector injection is preferred), the eye or the embryo with their corresponding transport barriers would usually altogether rely on topical delivery instead [286,287,288].

6.2. Traditional Viral and Non-Viral Vectors

The plethora of diverse ATMPs requires the use of different delivery vehicles and routes of administration of cells and genetic material for the achievement of optimal therapeutic effects in adults and children.
For the delivery of GTMPs, efficient transfer of genetic material or genome editing tools into target cells is a key step for success. For both ex vivo and in vivo applications, various viral and non-viral vectors have been developed as delivery vehicles, each one with their own advantages and disadvantages [289,290]. Improvements can be achieved by ongoing technical innovations, such as in the delivery of cells or genetic material to the tissues and cells of interest for in vivo application [291,292,293,294,295,296,297] or for ex vivo application in the isolation of suitable cells [16,298,299,300] or in more effective delivery into cells, and possibly nuclei, at improved efficiency and low toxicity [87,301,302,303,304,305,306]. Viral vectors (γ-retroviruses, lentiviruses, adenoviruses and adeno-associated viruses) were among the first exploited delivery platforms and are still highly prevalent due to their inherently high efficiency of gene transduction to eukaryotic cells (extensively reviewed in [106]). Here, AAVs in particular are almost universally exploited for in vivo application, whereas lentiviral vectors are usually the vectors of choice for permanent ex vivo HSPC modifications. Traditionally, ex vivo gene therapy has been applied to HSPCs, which is still associated with chemical myeloablation and corresponding treatment-related morbidities. Recent advances in antibody-mediated conditioning, such as through the targeting of CD117 cells, promise to improve the tolerability of HSPCs modified ex vivo and reduce treatment-related mortality [307]. Meanwhile, non-hematopoietic tissues are usually modified by in vivo strategies, which poses the problem of accessibility or homing to target tissues but has lower treatment-related morbidities. Lately, in vivo therapy is also being pursued for HSPCs as a safer, cheaper, and potentially more efficient therapeutic approach. Hemoglobinopathies, as the most common monogenic disorders, are once more paving the way as a test bed for new methodology, with the recent publication of several in vivo [293,308,309,310] and one in utero application of GTMPs [137]. Viral gene delivery is frequently associated with considerable immunogenicity and risks of genotoxicity, although many non-viral delivery methods have disadvantages of their own, including frequently lower transfer efficiency, and reduced specificity and duration of gene expression [311]. The fast-paced contemporary research field of non-viral vectors covers polymers, lipids, inorganic particles, engineered virus-like particles, hybrid systems of these vector types and naked nucleic acids for chemical or physical transfer [289,310,312]. For GTMPs, gene editing represents a special field of delivery application, because the persistence of editors would be detrimental and permanent changes in target cells can be introduced by highly transient action instead. The latter is most frequently achieved by electroporation ex vivo, whereas in vivo AAV-based delivery currently predominates [313], as a compromise between the desired efficiencies and the disadvantages of long persistence, low payload capacity and concerns over using high titers of viral vectors. Toward clinical application, there is, therefore, an increasing need for in vivo delivery technology with tissue specificity, flexible half-life, high payload capacity, reproducibility, GMP compliance and low immunogenicity. For traditional vectors, these properties are often difficult to achieve.

6.3. Nanomedicine

Nanomedicine has the potential to transform the delivery of therapeutic transgenes by providing highly versatile nanoparticle-based delivery platforms with improved safety profiles. Small particles in the nano-size range (at least one dimension < 100 nm [314]) can nowadays be engineered at large scales and with high precision to enable non-personalized as well as precision therapies [315]. Moreover, recent advances in nanoparticle designs to incorporate complex architectures, bio-response moieties and targeting agents allow for substantial control over their interactions with biological environments and help overcome biological barriers [315]. Therefore, nanoparticles can be tailored, among others, to protect the transgenes from degradation by nucleases, to reduce the stimulation of immune responses or to selectively target specific tissues or cell types to allow for maximum efficiency and minimal off-target effects. The genetic payload itself can be either entrapped into the nanoparticles or attached to the particle surface [316,317].
Side effects are of particular concern for early therapies in pregnancy, because the safety of the pregnant mother and the highly vulnerable developing fetus are at stake. In this context, the placenta, at the interface between maternal and fetal tissues, is critical for fetal development [318]. It governs active and passive gas, nutrient, hormone and waste transport, while blocking many larger molecules from passage to the fetus. Importantly, drug characteristics, such as acid/base properties, hydrophobicity or size, may affect selective passage and the potentially harmful accumulation of drugs in fetal, maternal or placental tissues. Nanoparticle-based delivery of therapeutics (e.g., chemical compounds, biologics and nucleic acids) may utilize such effects on placental translocation and interactions in order to facilitate the specific targeting of maternal, placental or fetal tissues for a highly targeted treatment and for the prevention of off-target effects [319]. For instance, the correction of maternal diseases will require a nanoparticle design that does not allow placental tissue accumulation or fetal translocation, whereas therapeutics for placental complications would rely on nanocarriers that preferentially locate to this particular organ. For fetal therapies, nanoparticles can be administered to the maternal circulation if particles with high placental transfer and specific targeting moieties for placental tissues can be identified. Alternatively, they can be injected directly into the amniotic fluid, umbilical vein or specific fetal tissues to bypass the placental barrier. However, even if the fetus is targeted directly, it will be important to ensure minimal fetal to maternal particle transfer. A recent study has proven the potential of in utero fetal gene editing by showing that the intra-amniotic administration of polymeric nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs was able to correct a disease-causing mutation in the β-globin gene in a mouse model of human β-thalassemia [175]. In another study, PLGA nanoparticles were used for efficient delivery of the CRISPR-complex (Cas9 protein, single gRNA and a fluorescent probe) into erythroid cells in vitro to elevate fetal globin expression [320]. Interestingly, the initial burst release of the content was followed by a sustained release pattern, indicating that intelligent nanocarrier designs could be further exploited to control for the release of the payload according to the therapeutic needs, e.g., to achieve fast release for genetic editing versus slow or sustained release for epigenetic or RNA editing.
In the past decade, significant efforts have been made to understand nanoparticle transport across the placenta in dependence of their physico-chemical properties and to identify targeting signals to direct their localization to specific tissues (mostly the placenta) [175]. Particle size is a key factor to affect placental translocation with a negative correlation (higher transfer for smaller particles), but other particle properties, such as surface charge, material composition or surface ligands, have an impact as well. Due to this complexity, it is still difficult to predict the placental transfer of a nanoparticle, and most likely a combination of multiple particle characteristics will determine its transplacental transport behavior. In addition to passive targeting approaches by the modulation of physico-chemical particle properties (e.g., size, charge, hydrophilicity, shape and chemical composition), active targeting strategies can enable the delivery of the payload to specific cell types or tissues. Several research teams have screened for and identified peptides or antibodies to target placental tissue [321,322]. For instance, Li et al. [323] have conjugated peptides targeting chondroitin sulfate A (CSA; expressed at the membrane of placental trophoblasts) to the surface of nanoparticles to deliver siNRF2 and sisFlt-1 to the placenta, which improved maternal and fetal outcomes in a preeclampsia mouse model [323]. Although siRNAs are not considered as ATMPs [324,325], this study highlights the feasibility of nanocarriers for the targeted placental delivery of genetic material to improve placental functions. In fact, proper placental function is essential for successful pregnancy, and consequently, placental dysfunction is involved in the pathogenesis of many pregnancy complications (e.g., intrauterine growth restriction, preeclampsia, preterm birth). In addition, recent work from Singh et al. indicates that persistent DNA damage in the placenta affects embryonic health, which emphasizes the importance of genome integrity for placental health and embryonic development [326]. Therefore, in addition to maternal or fetal therapy, the placenta could be an interesting target for ATMPs to improve health outcomes in complicated pregnancies and to reduce adverse health effects later in life.
Research on nanoformulations of chemical compounds, biologics or nucleic acids for in utero or pediatric use is still in its infancy, but slowly gathering momentum. In general, the main classes of nanoparticles exploited in nanomedicine applications are polymer-based, lipid-based, inorganic or dendrimer nanoparticles [315,327]. Most prenatal nanotherapies employ non-ATMP cargo, such as conventional drugs, siRNA and proteins [327,328], but there is growing interest to apply nanocarriers for gene addition and gene editing in pregnancy. In fact, the first transplacental gene delivery using plasmid DNA:lipopolyamine complexes to achieve non-invasive fetal drug delivery was reported as early as 1995 [329]. More recent examples explored transferrin-targeted PEGylated immunoliposomes to deliver plasmid DNA to fetal brain [330] or internalizing the arginine–glycine–aspartic acid (iRGD)-coated diblock copolymer complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1) to improve fetal growth restriction [331]. Although there are evidently tremendous opportunities for novel nanoparticle-based ATMPs, there are still some challenges ahead concerning the efficiency, stability and toxicity of nanocarriers [315]. These will need to be addressed comprehensively, such as by new intelligent nanoparticle designs, in order to achieve the full potential of nanoparticle-based delivery platforms and to establish safety for routine clinical application. In parallel, ongoing systematic assessments of patient safety, as well as of occupational and environmental risks along the life cycle of corresponding medicinal products and ATMPs, is required to share the information with all parties involved, including regulators, consultants, manufacturers, physicians and patients [332,333].
Systemic delivery via intravenous infusion is the most common approach for GTMP administration, although direct/local delivery of treatment into affected tissues (e.g., BM and the liver) is also used depending on clinical application [16]. Recently, the direct delivery to the fetal liver, lungs and intestines by the injection of mRNA-loaded LNPs into the fetal vitelline vein has been performed to achieve corresponding protein expression in the fetal liver [165]. For MSCs, both systemic delivery (intravenous/intraarterial infusions) or local/direct delivery (e.g., intramuscular and intratracheal injections) of cells have been tested in several preclinical and clinical studies [334]. In pediatric clinical trials of MSCs for aGvHD and bronchopulmonary dysplasia, intravenous and intratracheal routes are used, respectively [335,336], whereas for the limited in utero applications of MSCs for prenatal treatment of congenital diseases such as osteogenesis imperfecta and myelomeningocele, infusions via the umbilical vein or local/direct intraspinal infusions are administered [67,255].

7. Non-Technical Considerations for the Routine Application of Early ATMP Interventions

As technology, preclinical and clinical development has progressed to facilitate early interventions, a clear majority and a large proportion of the public express their approval of pediatric and of in utero applications of gene therapy to treat inherited diseases, respectively [337]. Beyond general attitudes, financial considerations clearly favor a shift from adult to early interventions for safe and efficacious treatments, whereas many ethical and regulatory impediments that remain for the still-developing ATMP sector (see Section 2) are further exacerbated, as detailed subsequently.

7.1. Financial Considerations

ATMPs are typically priced between USD 18,950 for tissue-engineered products and USD 1,206,751 for gene therapy, with the aforementioned prices excluding procurement, inventory and administration costs [338]. The high sales price of marketing-approved ATMPs can be attributed to a variety of factors, including cell sourcing (i.e., cell/tissue acquisition and expansion), GMP manufacturing (i.e., labor-, time- and cost-intensive GMP protocols and procedures, costly clinical-grade reagents and stringent quality control), distribution, and clinical application, including treatment and long-term follow-up [339]. The highly personalized nature of ATMPs, which restricts the scalability of manufacturing pipelines, and the small number of patients qualifying for these treatments, further add to the high prices of ATMPs. An examination of concrete cost factors specifically for ATMPs highlights the benefit of early interventions, in particular for the procurement of starting material and GMP manufacturing (including storage and distribution) and quality control (Figure 3).
The extraordinary cost of ATMPs compared with small-molecule drugs is most readily accepted where ATMPs represent potentially curative treatments, allowing financial comparisons of one-off cures vs. lifelong palliative treatments. However, for many ATMPs, there are additional, less tangible financial benefits. Even for ATMPs with the uncertainty of truly curative outcomes, considerations of permanently reduced disease severity for ATMP applications, of potentially catastrophic financial and health consequences for chronic palliative treatments, and of hope for a curative ATMP outcome or for prolonged survival and potential access to improved future therapies, should enter pricing considerations, in particular for ultra-rare diseases [198]. All three aspects strongly favor early intervention.
For the specific case of GTMPs, a major cost is that of vector production for the delivery of genetic materials. Here, juvenile or prenatal application would allow a ballpark change in materials, and thus, cost, per patient, in addition to reducing requirements for what is frequently limiting cell material for ex vivo GTMPs. Assuming an average body weight of 70 kg [340], a neonatal weight of 3.5 kg [341] and approximately 3 × 105 cells for in utero therapeutic intervention [137,342], an assumed vector cost of USD 100,000 per adult patient [89] at, e.g., 5 × 108 lentiviral transduction units per kg [194], would be reduced to USD 5000 in neonates and to USD 85 for in utero treatment [137,194,342]. Before any markup of commercial products and treatment-associated cost, this change in pricing would greatly increase accessibility of treatment.

7.2. Ethical and Regulatory Considerations

From the ethical and regulatory standpoint, de novo pediatric developments or adaptations of adult treatments are impaired by the absence of a consensus for the establishment of pediatric safety specifications, as analyzed elsewhere in this Special Issue [343]. Consequently, even for conventional small-molecule drugs, pediatric applications lag far behind developments for adults [344,345], as is also apparent in the TEDDY European Paediatric Medicines Database [346]. For ATMPs compared with conventional drugs, clinical studies on early interventions are at an additional disadvantage, because such studies frequently include long in-patient treatment in an unfamiliar environment and because of the difficulty of achieving truly informed consent for what are often highly sophisticated, hard-to-explain studies with many inherent uncertainties, both known and unknown [347]. Ethical issues generally prevail in pediatric drug studies, but do so even more in ATMP, and specifically GTMP studies. In contrast to other ATMPs, such as CAR-T cells developed to treat aggressive and otherwise lethal cancer types, non-toxic stem cells such as MSCs and autologous, genetically modified HSPCs are often envisioned to treat a variety of debilitating but manageable (if conventional supportive therapy is offered) genetic disorders, which substantially lowers the acceptable risk for corresponding drugs and greatly delays their development process, as well as their evaluation in children [348]. Specifically for HSPC-based GTMPs, concerns about drug-mediated insertional mutagenesis and off-targeting still remain the major hurdles for the endorsement of new pediatric clinical trials of related drugs, whereas for those drugs that make it through clinical trials, the need for the long-term monitoring of recipients for many years after drug administration sets back their final pediatric application approval [349,350,351]. In addition to these considerations for the general acceptability of ATMPs for early treatments, other concerns are uniquely associated with pediatric and/or in utero applications.

7.2.1. Pediatric

The same considerations that drive the long-established gap between the number of adult- and pediatric-approved conventional treatments, such as for small-molecule drugs, also drive the gap between adult and pediatric ATMP applications. For palliative treatments, this creates a dilemma where, in extreme cases, the treating physician may face the choice of unauthorized off-label use of adult medicines to pediatric patients [98], or of leaving the pediatric patient without treatment altogether. For often curative ATMPs, this dilemma is exacerbated, where pediatric patients, as far as has been analyzed, exhibit higher stem cell yields and fewer irreversible disease-related morbidities [194], and would be spared years of palliative treatment and reduced quality of life by early application. On the other side of the argument, safety considerations surrounding experimental treatments for underage patients create a strong counterincentive to trial the participation or approval of pediatric studies. As for adult studies, inclusion criteria for pediatric patients therefore invariably stipulate that trial participants do not respond to standard treatments, although what constitutes a satisfactory response and acceptable quality of life under standard treatments is often open to interpretation. Particularly for slowly progressing diseases, an additional consideration is the ongoing development and prospect of novel and potentially curative treatments, from which younger patients might still benefit later.

7.2.2. In Utero

At present, there is no legal framework for routine in utero ATMP application, chiefly due to the costly and technically demanding nature of the correspondingly limited body of preclinical work, combined with concerns about potential germline transmission and safety to mother and child [352]. Moreover, frequent uncertainty over genotype–phenotype correlation and the actual severity of the disease in postnatal life (e.g., due to genetic modifiers) may not allow clear-cut decisions based on medical necessity, and the relative certainty of a severe in utero or postnatal phenotype is a key criterion for in utero gene therapy according to the consensus statement by the International Fetal Transplantation and Immunology Society [352]. Correspondingly, better-established postnatal treatments are always preferred where disease onset and severity allow, in particular where it is feared that limited studies in large animal models may not have revealed all risks associated with prenatal treatment.
These points may weigh on the mind of bioethics review boards or of the treating physician, but they will also determine attitudes of the affected couples, because for in utero treatment, the notion of parental protection and responsibility is even more acute than for pediatric application. A combined feeling of responsibility, uncertainty over phenotype predictions and over the effectiveness and safety of treatment, and the option of postnatal treatment will combine to create a reluctance by parents to take up in utero therapy, if there are alternatives. After all, the condition might be manageable, or a catastrophic outcome of in utero treatment may come to burden them with the responsibility of having taken a wrong decision. Therefore, the trailblazers for in utero treatments are, and will be, the severest and earliest forms of genetic disease, where the risk–benefit ratio will more readily justify experimental treatments. Here, additional studies in large animal models will be needed to standardize in utero ATMP technologies and to shore up data in support of in utero treatment, as bases for the approval and development of corresponding clinical in utero ATMP applications.

8. Perspectives and Conclusions

Recent diagnostic and prognostic advances allow the ever-earlier informed application of ATMP products. More efficient, more affordable therapy is possible by in utero or pediatric applications, with vastly reduced cell and vector requirements for selected ATMP applications. In addition to improving affordability, efficiency and use of GMP resources, early application is fundamental to the treatment of many as-yet untreatable diseases with pre- or perinatal onset. There is, thus, every incentive for ATMPs to narrow the gap for pediatric vs. adult medication, aided by ongoing developments. Be it a growing number of in utero and pediatric studies, prolific research into improved cell isolation and expansion sources and technology, or continuing development of delivery technologies and versatile nanoparticles as vectors, conditions are shifting in favor of ATMPs and for their early application in particular. Critical work remains to address ethical and safety concerns for young or unborn patients, especially where data from adult studies are absent. However, as successful studies accumulate and establishment of underlying technologies and their ethical, regulatory and marketing framework conditions allows further development to gain momentum, prenatal and pediatric application of ATMPs promises safe, efficient and competitive treatments for a growing number of diseases and patients.
Resource, cost, efficiency and suitability advantages, helped by existing regulatory incentives for pediatric and orphan drug development and by a change in attitudes towards advanced therapies, disproportionately favor pediatric and in utero development for ATMPs, which holds the promise of an increase in the proportion of pediatric and early interventions in particular, and correspondingly earlier and better treatments in general.

Author Contributions

Resources, C.W.L., D.B. and M.K.; data curation, C.W.L. and L.K.; writing—original draft preparation, C.W.L., L.K., P.L.P., T.B.-T., S.L.G. and A.L.; writing—review and editing, C.W.L., L.K., P.L.P., T.B-T. and F.S.; visualization, C.W.L. and L.K.; supervision, C.W.L.; project administration, C.W.L.; funding acquisition, C.W.L., D.B. and M.K. All authors have read and agreed to the published version of the manuscript.


The research leading to these results has received funding from the European Union’s Horizon 2020 programme under Grant Agreement No. 777554. This work was co-financed by the European Regional Development Fund and the Republic of Cyprus through the Research and Innovation Foundation (Projects: EXCELLENCE/1216/0092, EXCELLENCE/0421/0086). The project “New infrastructure for diagnosis and treatment of patients” is funded by the Norway Grants 2014–2021.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

Not Applicable.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


  1. European Medicines Agency. Advanced Therapy Medicinal Products: Overview|European Medicines Agency. Available online: (accessed on 11 October 2021).
  2. European Medicines Agency. Advanced Therapy Classification|European Medicines Agency. Available online: (accessed on 11 October 2021).
  3. European Medicines Agency. Guidelines Relevant for Advanced Therapy Medicinal Products|European Medicines Agency. Available online: (accessed on 11 October 2021).
  4. European Medicines Agency. Marketing-Authorisation Procedures for Advanced-Therapy Medicinal Products|European Medicines Agency. Available online: (accessed on 11 October 2021).
  5. European Medicines Agency. Niraparib|Medicines|European Medicines Agency. Available online: (accessed on 11 October 2021).
  6. EC Medicines for Children|EC Public Health. Available online: (accessed on 11 October 2021).
  7. Halioua-Haubold, C.L.; Peyer, J.G.; Smith, J.A.; Arshad, Z.; Scholz, M.; Brindley, D.A.; Maclaren, R.E. Regulatory considerations for gene therapy products in the US, EU, and Japan. Yale J. Biol. Med. 2017, 90, 683–693. [Google Scholar] [PubMed]
  8. Iglesias-Lopez, C.; Obach, M.; Vallano, A.; Agustí, A. Comparison of regulatory pathways for the approval of advanced therapies in the European Union and the United States. Cytotherapy 2021, 23, 261–274. [Google Scholar] [CrossRef] [PubMed]
  9. Eder, C.; Wild, C. Technology forecast: Advanced therapies in late clinical research, EMA. approval or clinical application via hospital exemption. J. Mark. Access Health Policy 2019, 7, 1600939. [Google Scholar] [CrossRef] [PubMed]
  10. Gatline, A. Investor’s Business Daily® – Technology: Can CRISPR and These 3 Small Biotechs Cure 10,000 Diseases? Available online: (accessed on 17 May 2018).
  11. López-Paniagua, M.; de la Mata, A.; Galindo, S.; Blázquez, F.; Calonge, M.; Nieto-Miguel, T. Advanced Therapy Medicinal Products for the Eye: Definitions and Regulatory Framework. Pharmaceutics 2021, 13, 347. [Google Scholar] [CrossRef] [PubMed]
  12. Ronco, V.; Dilecce, M.; Lanati, E.; Canonico, P.L.; Jommi, C. Price and reimbursement of advanced therapeutic medicinal products in Europe: Are assessment and appraisal diverging from expert recommendations? J. Pharm. Policy Pract. 2021, 14, 30. [Google Scholar] [CrossRef]
  13. Mebarki, M.; Abadie, C.; Larghero, J.; Cras, A. Human umbilical cord-derived mesenchymal stem/stromal cells: A promising candidate for the development of advanced therapy medicinal products. Stem Cell Res. Ther. 2021, 12, 152. [Google Scholar] [CrossRef]
  14. Ciccocioppo, R.; Comoli, P.; Astori, G.; del Bufalo, F.; Prapa, M.; Dominici, M.; Locatelli, F. Developing cell therapies as drug products. Br. J. Pharmacol. 2021, 178, 262–279. [Google Scholar] [CrossRef]
  15. Elverum, K.; Whitman, M. Delivering cellular and gene therapies to patients: Solutions for realizing the potential of the next generation of medicine. Gene Ther. 2020, 27, 537–544. [Google Scholar] [CrossRef] [Green Version]
  16. Koniali, L.; Lederer, C.W.; Kleanthous, M. Therapy Development by Genome Editing of Hematopoietic Stem Cells. Cells 2021, 10, 1492. [Google Scholar] [CrossRef]
  17. Whomsley, R.; Palmi Reig, V.; Hidalgo-Simon, A. Environmental risk assessment of advanced therapies containing genetically modified organisms in the EU. Br. J. Clin. Pharmacol. 2021, 87, 2450–2458. [Google Scholar] [CrossRef]
  18. Lechanteur, C.; Briquet, A.; Bettonville, V.; Baudoux, E.; Beguin, Y. Msc manufacturing for academic clinical trials: From a clinical-grade to a full gmp-compliant process. Cells 2021, 10, 1320. [Google Scholar] [CrossRef] [PubMed]
  19. Beattie, S. Call for more effective regulation of clinical trials with advanced therapy medicinal products consisting of or containing genetically modified organisms in the European Union. Hum. Gene Ther. 2021, 32, 997–1003. [Google Scholar] [CrossRef] [PubMed]
  20. European Medicines Agency. Scientific Recommendations on Classification of Advanced Therapy Medicinal Products|EMA/140033/2021. Available online: (accessed on 10 December 2021).
  21. Attico, E.; Sceberras, V.; Pellegrini, G. Approaches for Effective Clinical Application of Stem Cell Transplantation. Curr. Transplant. Rep. 2018, 5, 244–250. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Quinn, C.; Young, C.; Thomas, J.; Trusheim, M. Estimating the Clinical Pipeline of Cell and Gene Therapies and Their Potential Economic Impact on the US Healthcare System. Value Health 2019, 22, 621–626. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Shukla, V.; Seoane-Vazquez, E.; Fawaz, S.; Brown, L.; Rodriguez-Monguio, R. The Landscape of Cellular and Gene Therapy Products: Authorization, Discontinuations, and Cost. Hum. Gene Ther. Clin. Dev. 2019, 30, 102–113. [Google Scholar] [CrossRef]
  24. Adami, A.; Maher, J. An overview of CAR T-cell clinical trial activity to 2021. Immunother. Adv. 2021, 1, ltab004. [Google Scholar] [CrossRef]
  25. Marofi, F.; Saleh, M.M.; Rahman, H.S.; Suksatan, W.; Al-Gazally, M.E.; Abdelbasset, W.K.; Thangavelu, L.; Yumashev, A.V.; Hassanzadeh, A.; Yazdanifar, M.; et al. CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies. Stem Cell Res. Ther. 2021, 12, 374. [Google Scholar] [CrossRef]
  26. Li, Y.-R.; Dunn, Z.S.; Zhou, Y.; Lee, D.; Yang, L. Development of Stem Cell-Derived Immune Cells for Off-the-Shelf Cancer Immunotherapies. Cells 2021, 10, 3497. [Google Scholar] [CrossRef]
  27. Aiuti, A.; Cattaneo, F.; Galimberti, S.; Benninghoff, U.; Cassani, B.; Callegaro, L.; Scaramuzza, S.; Andolfi, G.; Mirolo, M.; Brigida, I.; et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 2009, 360, 447–458. [Google Scholar] [CrossRef] [Green Version]
  28. Bank, A.; Dorazio, R.; Leboulch, P. A phase I/II clinical trial of beta-globin gene therapy for beta-thalassemia. Ann. N. Y. Acad. Sci. 2005, 1054, 308–316. [Google Scholar] [CrossRef]
  29. Cavazzana-Calvo, M.; Payen, E.; Negre, O.; Wang, G.; Hehir, K.; Fusil, F.; Down, J.; Denaro, M.; Brady, T.; Westerman, K.; et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 2010, 467, 318–322. [Google Scholar] [CrossRef] [PubMed]
  30. Ribeil, J.-A.; Hacein-Bey-Abina, S.; Payen, E.; Magnani, A.; Semeraro, M.; Magrin, E.; Caccavelli, L.; Neven, B.; Bourget, P.; El Nemer, W.; et al. Gene Therapy in a Patient with Sickle Cell Disease. N. Engl. J. Med. 2017, 376, 848–855. [Google Scholar] [CrossRef] [PubMed]
  31. Thompson, A.A.; Walters, M.C.; Kwiatkowski, J.; Rasko, J.E.J.; Ribeil, J.-A.A.; Hongeng, S.; Magrin, E.; Schiller, G.J.; Payen, E.; Semeraro, M.; et al. Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia. N. Engl. J. Med. 2018, 378, 1479–1493. [Google Scholar] [CrossRef] [PubMed]
  32. CRISPRTX. CRISPR Therapeutics Provides Business Update and Reports Fourth Quarter and Full Year 2020 Financial Results. Available online: (accessed on 1 June 2021).
  33. Gruhn, B.; Brodt, G.; Ernst, J. Extended Treatment with Mesenchymal Stromal Cells-Frankfurt am Main in a Pediatric Patient with Steroid-refractory Acute Gastrointestinal Graft-Versus-Host Disease: Case Report and Review of the Literature. J. Pediatr. Hematol. Oncol. 2021, 43, e419–e425. [Google Scholar] [CrossRef] [PubMed]
  34. Buscail, E.; Le Cosquer, G.; Gross, F.; Lebrin, M.; Bugarel, L.; Deraison, C.; Vergnolle, N.; Bournet, B.; Gilletta, C.; Buscail, L. Adipose-derived stem cells in the treatment of perianal fistulas in Crohn’s disease: Rationale, clinical results and perspectives. Int. J. Mol. Sci. 2021, 22, 9967. [Google Scholar] [CrossRef]
  35. Cuende, N.; Rasko, J.E.J.; Koh, M.B.C.; Dominici, M.; Ikonomou, L. Cell, tissue and gene products with marketing authorization in 2018 worldwide. Cytotherapy 2018, 20, 1401–1413. [Google Scholar] [CrossRef]
  36. Globerson Levin, A.; Rivière, I.; Eshhar, Z.; Sadelain, M. CAR T cells: Building on the CD19 paradigm. Eur. J. Immunol. 2021, 51, 2151–2163. [Google Scholar] [CrossRef]
  37. Lee, D.W.; Kochenderfer, J.N.; Stetler-Stevenson, M.; Cui, Y.K.; Delbrook, C.; Feldman, S.A.; Fry, T.J.; Orentas, R.; Sabatino, M.; Shah, N.N.; et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: A phase 1 dose-escalation trial. Lancet 2015, 385, 517–528. [Google Scholar] [CrossRef]
  38. Lichtenstein, D.A.; Schischlik, F.; Shao, L.; Steinberg, S.M.; Yates, B.; Wang, H.W.; Wang, Y.; Inglefield, J.; Dulau-Florea, A.; Ceppi, F.; et al. Characterization of HLH-like manifestations as a CRS variant in patients receiving CD22 CAR T cells. Blood 2021, 138, 2469–2484. [Google Scholar] [CrossRef]
  39. Liu, R.; Cheng, Q.; Kang, L.; Wang, E.; Li, Y.; Zhang, J.; Xiao, H.; Zhang, Y.; Chu, L.; Chen, X.; et al. CD19 or CD20 CAR T-cell Therapy Demonstrates Durable Antitumor Efficacy in Patients with CNS Lymphoma. Hum. Gene Ther. 2022, 33, 318–329. [Google Scholar] [CrossRef]
  40. Shah, N.; Chari, A.; Scott, E.; Mezzi, K.; Usmani, S.Z. B-cell maturation antigen (BCMA) in multiple myeloma: Rationale for targeting and current therapeutic approaches. Leukemia 2020, 34, 985–1005. [Google Scholar] [CrossRef] [PubMed]
  41. Xue, Y.B.; Lai, X.; Li, R.L.; Ge, C.L.; Zeng, B.Z.; Li, Z.; Fu, Q.F.; Zhao, L.F.; Dong, S.W.; Yang, J.Y.; et al. CD19 and CD30 CAR T-Cell Immunotherapy for High-Risk Classical Hodgkin’s Lymphoma. Front. Oncol. 2021, 10, 607362. [Google Scholar] [CrossRef] [PubMed]
  42. Zhang, H.; Liu, M.; Xiao, X.; Lv, H.; Jiang, Y.; Li, X.; Yuan, T.; Zhao, M. A combination of humanized anti-BCMA and murine anti-CD38 CAR-T cell therapy in patients with relapsed or refractory multiple myeloma. Leuk. Lymphoma 2022, 1–10. [Google Scholar] [CrossRef] [PubMed]
  43. Mohanty, R.; Chowdhury, C.R.; Arega, S.; Sen, P.; Ganguly, P.; Ganguly, N. CAR T cell therapy: A new era for cancer treatment (Review). Oncol. Rep. 2019, 42, 2183–2195. [Google Scholar] [CrossRef]
  44. Tang, Y.; Yin, H.; Zhao, X.; Jin, D.; Liang, Y.; Xiong, T.; Li, L.; Tang, W.; Zhang, J.; Liu, M.; et al. High efficacy and safety of CD38 and BCMA bispecific CAR-T in relapsed or refractory multiple myeloma. J. Exp. Clin. Cancer Res. 2022, 41, 1–15. [Google Scholar] [CrossRef]
  45. Shah, N.N.; Johnson, B.D.; Schneider, D.; Zhu, F.; Szabo, A.; Keever-Taylor, C.A.; Krueger, W.; Worden, A.A.; Kadan, M.J.; Yim, S.; et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: A phase 1 dose escalation and expansion trial. Nat. Med. 2020, 26, 1569–1575. [Google Scholar] [CrossRef]
  46. Xie, G.; Dong, H.; Liang, Y.; Ham, J.D.; Rizwan, R.; Chen, J. CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine 2020, 59, 102975. [Google Scholar] [CrossRef]
  47. Tucci, F.; Scaramuzza, S.; Aiuti, A.; Mortellaro, A. Update on Clinical Ex Vivo Hematopoietic Stem Cell Gene Therapy for Inherited Monogenic Diseases. Mol. Ther. 2021, 29, 489–504. [Google Scholar] [CrossRef]
  48. Simaria, A.S.; Hassan, S.; Varadaraju, H.; Rowley, J.; Warren, K.; Vanek, P.; Farid, S.S. Allogeneic cell therapy bioprocess economics and optimization: Single-use cell expansion technologies. Biotechnol. Bioeng. 2014, 111, 69–83. [Google Scholar] [CrossRef] [Green Version]
  49. Papasavva, P.; Kleanthous, M.; Lederer, C.W. Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases. Mol. Diagn. Ther. 2019, 23, 201–222. [Google Scholar] [CrossRef] [Green Version]
  50. Papanikolaou, E.; Bosio, A. The Promise and the Hope of Gene Therapy. Front. Genome Ed. 2021, 3, 618346. [Google Scholar] [CrossRef] [PubMed]
  51. European Medicines Agency. Zynteglo. Available online: (accessed on 21 March 2022).
  52. European Medicines Agency. Skysona. Available online: (accessed on 21 March 2022).
  53. European Medicines Agency. Strimvelis. Available online: (accessed on 21 March 2022).
  54. European Medicines Agency. Libmeldy. Available online: (accessed on 21 March 2022).
  55. European Medicines Agency. Luxturna. Available online: (accessed on 21 March 2022).
  56. European Medicines Agency. Zolgensma. Available online: (accessed on 21 March 2022).
  57. European Medicines Agency. Resamirigene Bilparvovec. Available online: (accessed on 21 March 2022).
  58. Lazarus, H.M.; Haynesworth, S.E.; Gerson, S.L.; Rosenthal, N.S.; Caplan, A.I. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): Implications for therapeutic use. Bone Marrow Transplant. 1995, 16, 557–564. [Google Scholar] [PubMed]
  59. Jacobsohn, D.A.; Vogelsang, G.B. Acute graft versus host disease. Orphanet J. Rare Dis. 2007, 2, 35. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Mac Sweeney, R.; McAuley, D.F. Mesenchymal stem cell therapy in acute lung injury: Is it time for a clinical trial? Thorax 2012, 67, 475–476. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Aggarwal, S.; Pittenger, M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005, 105, 1815–1822. [Google Scholar] [CrossRef] [Green Version]
  62. Maitra, B.; Szekely, E.; Gjini, K.; Laughlin, M.J.; Dennis, J.; Haynesworth, S.E.; Koç, O.N. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant. 2004, 33, 597–604. [Google Scholar] [CrossRef] [Green Version]
  63. Introna, M.; Lucchini, G.; Dander, E.; Galimberti, S.; Rovelli, A.; Balduzzi, A.; Longoni, D.; Pavan, F.; Masciocchi, F.; Algarotti, A.; et al. Treatment of graft versus host disease with mesenchymal stromal cells: A phase I study on 40 adult and pediatric patients. Biol. Blood Marrow Transplant. 2014, 20, 375–381. [Google Scholar] [CrossRef] [Green Version]
  64. Le Blanc, K.; Frassoni, F.; Ball, L.; Locatelli, F.; Roelofs, H.; Lewis, I.; Lanino, E.; Sundberg, B.; Bernardo, M.E.; Remberger, M.; et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: A phase II study. Lancet 2008, 371, 1579–1586. [Google Scholar] [CrossRef]
  65. McIntyre, L.A.; Moher, D.; Fergusson, D.A.; Sullivan, K.J.; Mei, S.H.J.; Lalu, M.; Marshall, J.; McLeod, M.; Griffin, G.; Grimshaw, J.; et al. Efficacy of mesenchymal stromal cell therapy for acute lung injury in preclinical animal models: A systematic review. PLoS ONE 2016, 11, e0147170. [Google Scholar] [CrossRef] [Green Version]
  66. Rojas, M.; Xu, J.; Woods, C.R.; Mora, A.L.; Spears, W.; Roman, J.; Brigham, K.L. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am. J. Respir. Cell Mol. Biol. 2005, 33, 145–152. [Google Scholar] [CrossRef] [Green Version]
  67. Sagar, R.; David, A.; Gotherstrom, C. BOOSTB4 (Boost Brittle Bones before Birth) trial protocol. Prenat. Diagn. 2020, 40, 46. [Google Scholar]
  68. Otsuru, S.; Desbourdes, L.; Guess, A.J.; Hofmann, T.J.; Relation, T.; Kaito, T.; Dominici, M.; Iwamoto, M.; Horwitz, E.M. Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta. Cytotherapy 2018, 20, 62–73. [Google Scholar] [CrossRef] [PubMed]
  69. Dong, R.; Bai, Y.; Dai, J.; Deng, M.; Zhao, C.; Tian, Z.; Zeng, F.; Liang, W.; Liu, L.; Dong, S. Engineered scaffolds based on mesenchymal stem cells/preosteoclasts extracellular matrix promote bone regeneration. J. Tissue Eng. 2020, 11, 1–13. [Google Scholar] [CrossRef] [PubMed]
  70. Kon, E.; Muraglia, A.; Corsi, A.; Bianco, P.; Marcacci, M.; Martin, I.; Boyde, A.; Ruspantini, I.; Chistolini, P.; Rocca, M.; et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J. Biomed. Mater. Res. 2000, 49, 328–337. [Google Scholar] [CrossRef]
  71. Van Gaalen, S.M.; Dhert, W.J.A.; Van Den Muysenberg, A.; Oner, F.C.; Van Blitterswijk, C.; Verbout, A.J.; De Bruijn, J.D. Bone Tissue Engineering for Spine Fusion: An Experimental Study on Ectopic and Orthotopic Implants in Rats. Tissue Eng. 2004, 10, 231–239. [Google Scholar] [CrossRef]
  72. Vicinanza, C.; Lombardi, E.; Da Ros, F.; Marangon, M.; Durante, C.; Mazzucato, M.; Agostini, F. Modified mesenchymal stem cells in cancer therapy: A smart weapon requiring upgrades for wider clinical applications. World J. Stem Cells 2022, 14, 54–75. [Google Scholar] [CrossRef]
  73. Harrell, C.R.; Volarevic, A.; Djonov, V.G.; Jovicic, N.; Volarevic, V. Mesenchymal stem cell: A friend or foe in anti-tumor immunity. Int. J. Mol. Sci. 2021, 22, 12429. [Google Scholar] [CrossRef]
  74. Premer, C.; Blum, A.; Bellio, M.A.; Schulman, I.H.; Hurwitz, B.E.; Parker, M.; Dermarkarian, C.R.; DiFede, D.L.; Balkan, W.; Khan, A.; et al. Allogeneic Mesenchymal Stem Cells Restore Endothelial Function in Heart Failure by Stimulating Endothelial Progenitor Cells. EBioMedicine 2015, 2, 467–475. [Google Scholar] [CrossRef] [Green Version]
  75. Liu, M.; He, J.; Zheng, S.; Zhang, K.; Ouyang, Y.; Zhang, Y.; Li, C.; Wu, D. Human umbilical cord mesenchymal stem cells ameliorate acute liver failure by inhibiting apoptosis, inflammation and pyroptosis. Ann. Transl. Med. 2021, 9, 1615. [Google Scholar] [CrossRef]
  76. Liu, Q.; Lv, C.; Jiang, Y.; Luo, K.; Gao, Y.; Liu, J.; Zhang, X.; Mohammad Omar, J.; Jin, S. From hair to liver: Emerging application of hair follicle mesenchymal stem cell transplantation reverses liver cirrhosis by blocking the TGF-β/Smad signaling pathway to inhibit pathological HSC activation. PeerJ. 2022, 10, e12872. [Google Scholar] [CrossRef]
  77. Huang, Y.; Yang, L. Mesenchymal stem cells and extracellular vesicles in therapy against kidney diseases. Stem Cell Res. Ther. 2021, 12, 219. [Google Scholar] [CrossRef] [PubMed]
  78. Mastrolia, I.; Foppiani, E.M.; Murgia, A.; Candini, O.; Samarelli, A.V.; Grisendi, G.; Veronesi, E.; Horwitz, E.M.; Dominici, M. Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review. Stem Cells Transl. Med. 2019, 8, 1135–1148. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Sherkow, J.S. Controlling CRISPR Through Law: Legal Regimes as Precautionary Principles. Cris. J. 2019, 2, 299–303. [Google Scholar] [CrossRef] [PubMed]
  80. Daley, G.Q.; Lovell-Badge, R.; Steffann, J. After the Storm—A Responsible Path for Genome Editing. N. Engl. J. Med. 2019, 380, 897–899. [Google Scholar] [CrossRef] [Green Version]
  81. Cockroft, A.; Wilson, A. Comparability: What we can learn from the review of advanced therapy medicinal products. Regen. Med. 2021, 16, 655–667. [Google Scholar] [CrossRef]
  82. Coppens, D.G.M.; de Wilde, S.; Guchelaar, H.J.; De Bruin, M.L.; Leufkens, H.G.M.; Meij, P.; Hoekman, J. A decade of marketing approval of gene and cell-based therapies in the United States, European Union and Japan: An evaluation of regulatory decision-making. Cytotherapy 2018, 20, 769–778. [Google Scholar] [CrossRef]
  83. Gozzo, L.; Romano, G.L.; Romano, F.; Brancati, S.; Longo, L.; Vitale, D.C.; Drago, F. Health Technology Assessment of Advanced Therapy Medicinal Products: Comparison Among 3 European Countries. Front. Pharmacol. 2021, 12, 755052. [Google Scholar] [CrossRef]
  84. Ten Ham, R.M.T.; Hoekman, J.; Hövels, A.M.; Broekmans, A.W.; Leufkens, H.G.M.; Klungel, O.H. Challenges in Advanced Therapy Medicinal Product Development: A Survey among Companies in Europe. Mol. Ther. Methods Clin. Dev. 2018, 11, 121–130. [Google Scholar] [CrossRef] [Green Version]
  85. Adair, J.; Sevilla, J.; Heredia, C.; Becker, P.; Kiem, H.-P.; Bueren, J. Lessons Learned from Two Decades of Clinical Trial Experience in Gene Therapy for Fanconi Anemia. Curr. Gene Ther. 2017, 16, 338–348. [Google Scholar] [CrossRef]
  86. Jossen, V.; Muoio, F.; Panella, S.; Harder, Y.; Tallone, T.; Eibl, R. An approach towards a gmp compliant in-vitro expansion of human adipose stem cells for autologous therapies. Bioengineering 2020, 7, 77. [Google Scholar] [CrossRef]
  87. Agostini, F.; Vicinanza, C.; Biolo, G.; Spessotto, P.; Da Ros, F.; Lombardi, E.; Durante, C.; Mazzucato, M. Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications. Cells 2021, 10, 3412. [Google Scholar] [CrossRef]
  88. Ayati, N.; Saiyarsarai, P.; Nikfar, S. Short and long term impacts of COVID-19 on the pharmaceutical sector. DARU J. Pharm. Sci. 2020, 28, 799–805. [Google Scholar] [CrossRef] [PubMed]
  89. Plieth, J. The $100,000 Problem Gene Therapy Companies Would Rather Not Mention|Evaluate. Available online: (accessed on 31 December 2021).
  90. Gonçalves, E. Advanced therapy medicinal products: Value judgement and ethical evaluation in health technology assessment. Eur. J. Health Econ. 2020, 21, 311–320. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  91. BioPharma Dive. Available online: (accessed on 18 May 2021).
  92. Rackaityte, E.; Halkias, J. Mechanisms of Fetal T Cell Tolerance and Immune Regulation. Front. Immunol. 2020, 11, 588. [Google Scholar] [CrossRef]
  93. Bose, S.K.; Menon, P.; Peranteau, W.H. In Utero Gene Therapy: Progress and Challenges. Trends Mol. Med. 2021, 27, 728–730. [Google Scholar] [CrossRef]
  94. European Medicines Agency. Paediatric Regulation|European Medicines Agency. Available online: (accessed on 11 October 2021).
  95. EUR-Lex-32006R1901-EN-EUR-Lex. Available online: (accessed on 24 February 2022).
  96. EUR-Lex-32006R1902-EN-EUR-Lex. Available online: (accessed on 24 February 2022).
  97. European Commission. State of Paediatric Medicines in the EU – 10 Years of the EU Paediatric Regulation; COM(2017)626; European Commission: Brussel, Belgium, 2017. [Google Scholar]
  98. European Medicines Agency. 10-Year Report to the European Commission – General Report on the Paediatric Regulation; EMA/231225/2015; European Medicines Agency: Amsterdam, The Netherlands, 2017. [Google Scholar]
  99. Park, P.J.; Colletti, E.; Ozturk, F.; Wood, J.A.; Tellez, J.; Almeida-Porada, G.; Porada, C.D. Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep. Hum. Gene Ther. 2009, 20, 201–215. [Google Scholar] [CrossRef] [PubMed]
  100. Almeida-Porada, G.; Atala, A.; Porada, C.D. In utero stem cell transplantation and gene therapy: Rationale, history, and recent advances toward clinical application. Mol. Ther.-Methods Clin. Dev. 2016, 3, 16020. [Google Scholar] [CrossRef] [Green Version]
  101. Staud, F.; Karahoda, R. Trophoblast: The central unit of fetal growth, protection and programming. Int. J. Biochem. Cell Biol. 2018, 105, 35–40. [Google Scholar] [CrossRef]
  102. Sharma, A.; Sah, N.; Kannan, S.; Kannan, R.M. Targeted drug delivery for maternal and perinatal health: Challenges and opportunities. Adv. Drug Deliv. Rev. 2021, 177, 113950. [Google Scholar] [CrossRef]
  103. Schrepfer, S.; Deuse, T.; Reichenspurner, H.; Fischbein, M.P.; Robbins, R.C.; Pelletier, M.P. Stem Cell Transplantation: The Lung Barrier. Transplant. Proc. 2007, 39, 573–576. [Google Scholar] [CrossRef]
  104. Nijagal, A.; Le, T.; Wegorzewska, M.; MacKenzie, T.C. A mouse model of in Utero transplantation. J. Vis. Exp. 2010, e2303. [Google Scholar] [CrossRef] [Green Version]
  105. Mattar, C.N.Z.; Gil-Farina, I.; Rosales, C.; Johana, N.; Tan, Y.Y.W.; McIntosh, J.; Kaeppel, C.; Waddington, S.N.; Biswas, A.; Choolani, M.; et al. In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model. Mol. Ther. 2017, 25, 1843–1853. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Palanki, R.; Peranteau, W.H.; Mitchell, M.J. Delivery technologies for in utero gene therapy. Adv. Drug Deliv. Rev. 2021, 169, 51–62. [Google Scholar] [CrossRef] [PubMed]
  107. European Medicines Agency. EMEA/273974/2005-Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products. Available online: (accessed on 8 November 2018).
  108. European Commission. Guidelines on Good Clinical Practice specific to Advanced Therapy Medicinal Products; C(2019)7140; European Commission: Brussel, Belgium, 2019. [Google Scholar]
  109. Jaeggi, E.T.; Carvalho, J.S.; De Groot, E.; Api, O.; Clur, S.A.B.; Rammeloo, L.; McCrindle, B.W.; Ryan, G.; Manlhiot, C.; Blom, N.A. Comparison of transplacental treatment of fetal supraventricular tachyarrhythmias with digoxin, flecainide, and sotalol: Results of a nonrandomized multicenter study. Circulation 2011, 124, 1747–1754. [Google Scholar] [CrossRef] [Green Version]
  110. Cerveny, L.; Murthi, P.; Staud, F. HIV in pregnancy: Mother-to-child transmission, pharmacotherapy, and toxicity. Biochim. Biophys. Acta-Mol. Basis Dis. 2021, 1867, 166206. [Google Scholar] [CrossRef]
  111. Korth-Bradley, J.M. The Path to Perfect Pediatric Posology—Drug Development in Pediatrics. J. Clin. Pharmacol. 2018, 58, S48–S57. [Google Scholar] [CrossRef] [Green Version]
  112. Manual, R.; Ray, L. Breeding Strategies for Maintaining Colonies of Laboratory Mice: A Jackson Laboratory Resource Manual; The Jackson Laboratory: Bar Harbor, ME, USA, 2007; Volume 83, p. 29. [Google Scholar]
  113. Ayuso, M.; Buyssens, L.; Stroe, M.; Valenzuela, A.; Allegaert, K.; Smits, A.; Annaert, P.; Mulder, A.; Carpentier, S.; Van Ginneken, C.; et al. The neonatal and juvenile pig in pediatric drug discovery and development. Pharmaceutics 2021, 13, 44. [Google Scholar] [CrossRef]
  114. Trobridge, G.D.; Kiem, H.P. Large animal models of hematopoietic stem cell gene therapy. Gene Ther. 2010, 17, 939–948. [Google Scholar] [CrossRef] [Green Version]
  115. Story, B.D.; Miller, M.E.; Bradbury, A.M.; Million, E.D.; Duan, D.; Taghian, T.; Faissler, D.; Fernau, D.; Beecy, S.J.; Gray-Edwards, H.L. Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases. Front. Vet. Sci. 2020, 7, 80. [Google Scholar] [CrossRef] [Green Version]
  116. Chen, Y.; Niu, Y.; Ji, W. Genome editing in nonhuman primates: Approach to generating human disease models. J. Intern. Med. 2016, 280, 246–251. [Google Scholar] [CrossRef]
  117. Sasaki, E.; Sakakibara, Y.; Kumita, W.; Ito, R.; Nozu, R.; Inoue, T.; Katano, I.; Okahara, N.; Okahara, J.; Shimizu, Y.; et al. Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing. Cell Stem Cell 2016, 19, 127–138. [Google Scholar] [CrossRef] [Green Version]
  118. Gray-Edwards, H.L.; Randle, A.N.; Maitland, S.A.; Benatti, H.R.; Hubbard, S.M.; Canning, P.F.; Vogel, M.B.; Brunson, B.L.; Hwang, M.; Ellis, L.E.; et al. Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease. Hum. Gene Ther. 2018, 29, 312–326. [Google Scholar] [CrossRef] [PubMed]
  119. Kleine Holthaus, S.M.; Aristorena, M.; Maswood, R.; Semenyuk, O.; Hoke, J.; Hare, A.; Smith, A.J.; Mole, S.E.; Ali, R.R. Gene Therapy Targeting the Inner Retina Rescues the Retinal Phenotype in a Mouse Model of CLN3 Batten Disease. Hum. Gene Ther. 2020, 31, 709–718. [Google Scholar] [CrossRef] [PubMed]
  120. Song, C.; Dufour, V.L.; Cideciyan, A.V.; Ye, G.J.; Swider, M.; Newmark, J.A.; Timmers, A.M.; Robinson, P.M.; Knop, D.R.; Chulay, J.D.; et al. Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy. Hum. Gene Ther. 2020, 31, 743–755. [Google Scholar] [CrossRef]
  121. Hastings, M.L.; Brigande, J.V. Fetal gene therapy and pharmacotherapy to treat congenital hearing loss and vestibular dysfunction. Hear. Res. 2020, 394, 107931. [Google Scholar] [CrossRef]
  122. Fåne, A.; Myhre, M.R.; Inderberg, E.M.; Wälchli, S. In vivo experimental mouse model to test CD19CAR T cells generated with different methods. Methods Cell Biol. 2022, 167, 149–161. [Google Scholar] [CrossRef]
  123. Wang, Y.; Buck, A.; Grimaud, M.; Culhane, A.C.; Kodangattil, S.; Razimbaud, C.; Bonal, D.M.; De Nguyen, Q.; Zhu, Z.; Wei, K.; et al. Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model. Mol. Ther.-Oncolytics 2022, 24, 385–399. [Google Scholar] [CrossRef]
  124. Daher, M.; Basar, R.; Gokdemir, E.; Baran, N.; Uprety, N.; Nunez Cortes, A.K.; Mendt, M.; Kerbauy, L.N.; Banerjee, P.P.; Shanley, M.; et al. Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells. Blood 2021, 137, 624–636. [Google Scholar] [CrossRef]
  125. Makkouk, A.; Yang, X.C.; Barca, T.; Lucas, A.; Turkoz, M.; Wong, J.T.S.; Nishimoto, K.P.; Brodey, M.M.; Tabrizizad, M.; Gundurao, S.R.Y.; et al. Off-the-shelf Vδ 1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma. J. Immunother. Cancer 2021, 9, e003441. [Google Scholar] [CrossRef]
  126. Goyama, S.; Wunderlich, M.; Mulloy, J.C. Xenograft models for normal and malignant stem cells. Blood 2015, 125, 2630–2640. [Google Scholar] [CrossRef] [Green Version]
  127. Radtke, S.; Humbert, O.; Kiem, H.P. Mouse models in hematopoietic stem cell gene therapy and genome editing. Biochem. Pharmacol. 2020, 174, 113692. [Google Scholar] [CrossRef] [PubMed]
  128. Mortellaro, A.; Hernandez, R.J.; Guerrini, M.M.; Carlucci, F.; Tabucchi, A.; Ponzoni, M.; Sanvito, F.; Doglioni, C.; Di Serio, C.; Biasco, L.; et al. Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects. Blood 2006, 108, 2979–2988. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  129. Young, H.W.J.; Molina, J.G.; Dimina, D.; Zhong, H.; Jacobson, M.; Chan, L.-N.L.; Chan, T.-S.; Lee, J.J.; Blackburn, M.R. A 3 Adenosine Receptor Signaling Contributes to Airway Inflammation and Mucus Production in Adenosine Deaminase-Deficient Mice. J. Immunol. 2004, 173, 1380–1389. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  130. Blackburn, M.R.; Datta, S.K.; Kellems, R.E. Adenosine deaminase-deficient mice generated using a two-stage genetic engineering strategy exhibit a combined immunodeficiency. J. Biol. Chem. 1998, 273, 5093–5100. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  131. Walia, J.S.; Altaleb, N.; Bello, A.; Kruck, C.; LaFave, M.C.; Varshney, G.K.; Burgess, S.M.; Chowdhury, B.; Hurlbut, D.; Hemming, R.; et al. Long-term correction of Sandhoff disease following intravenous delivery of rAAV9 to mouse neonates. Mol. Ther. 2015, 23, 414–422. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  132. Massaro, G.; Geard, A.F.; Liu, W.; Coombe-tennant, O.; Waddington, S.N.; Baruteau, J.; Gissen, P.; Rahim, A.A. Gene therapy for lysosomal storage disorders: Ongoing studies and clinical development. Biomolecules 2021, 11, 611. [Google Scholar] [CrossRef] [PubMed]
  133. Scaramuzza, S.; Biasco, L.; Ripamonti, A.; Castiello, M.C.; Loperfido, M.; Draghici, E.; Hernandez, R.J.; Benedicenti, F.; Radrizzani, M.; Salomoni, M.; et al. Preclinical Safety and Efficacy of human CD34 + Cells transduced with lentiviral vector for the treatment of wiskott-aldrich syndrome. Mol. Ther. 2013, 21, 175–184. [Google Scholar] [CrossRef] [Green Version]
  134. Huo, Y.; McConnell, S.C.; Liu, S.; Zhang, T.; Yang, R.; Ren, J.; Ryan, T.M. Humanized mouse models of Cooley’s anemia: Correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology. Ann. N. Y. Acad. Sci. 2010, 1202, 45–51. [Google Scholar] [CrossRef]
  135. Huo, Y.; McConnell, S.C.; Liu, S.R.; Yang, R.; Zhang, T.T.; Sun, C.W.; Wu, L.C.; Ryan, T.M. Humanized Mouse Model of Cooley’s Anemia. J. Biol. Chem. 2009, 284, 4889–4896. [Google Scholar] [CrossRef] [Green Version]
  136. Ciavatta, D.J.; Ryan, T.M.; Farmer, S.C.; Townes, T.M. Mouse model of human beta zero thalassemia: Targeted deletion of the mouse beta maj-and beta min-globin genes in embryonic stem cells. Proc. Natl. Acad. Sci. USA 1995, 92, 9259–9263. [Google Scholar] [CrossRef] [Green Version]
  137. Shangaris, P.; Loukogeorgakis, S.P.; Subramaniam, S.; Flouri, C.; Jackson, L.H.; Wang, W.; Blundell, M.P.; Liu, S.; Eaton, S.; Bakhamis, N.; et al. In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques. Sci. Rep. 2019, 9, 11592. [Google Scholar] [CrossRef] [PubMed]
  138. Huo, Y.; McConnell, S.C.; Ryan, T.M. Preclinical transfusion-dependent humanized mouse model of beta thalassemia major. Blood 2009, 113, 4763–4770. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  139. Casal, M.; Haskins, M. Large animal models and gene therapy. Eur. J. Hum. Genet. 2006, 14, 266–272. [Google Scholar] [CrossRef] [PubMed]
  140. Chinnadurai, R.; Ng, S.; Velu, V.; Galipeau, J. Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease. World J. Gastroenterol. 2015, 21, 4779–4787. [Google Scholar] [CrossRef]
  141. Chinnadurai, R.; Garcia, M.A.; Sakurai, Y.; Lam, W.A.; Kirk, A.D.; Galipeau, J.; Copland, I.B. Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo. Stem Cell Rep. 2014, 3, 60–72. [Google Scholar] [CrossRef] [Green Version]
  142. Lee, H.K.; Lim, S.H.; Chung, I.S.; Park, Y.; Park, M.J.; Kim, J.Y.; Kim, Y.G.; Hong, J.T.; Kim, Y.; Han, S.-B. Preclinical Efficacy and Mechanisms of Mesenchymal Stem Cells in Animal Models of Autoimmune Diseases. Immune Netw. 2014, 14, 81–88. [Google Scholar] [CrossRef] [Green Version]
  143. Lu, S.; Zhu, K.; Guo, Y.; Wang, E.; Huang, J. Evaluation of animal models of Crohn’s disease with anal fistula (Review). Exp. Ther. Med. 2021, 22, 974. [Google Scholar] [CrossRef]
  144. Harman, R.M.; Marx, C.; Van de Walle, G.R. Translational Animal Models Provide Insight Into Mesenchymal Stromal Cell (MSC) Secretome Therapy. Front. Cell Dev. Biol. 2021, 9, 654885. [Google Scholar] [CrossRef]
  145. Hou, H.; Zhang, L.; Duan, L.; Liu, Y.; Han, Z.; Li, Z.; Cao, X. Spatio-Temporal Metabolokinetics and Efficacy of Human Placenta-Derived Mesenchymal Stem/Stromal Cells on Mice with Refractory Crohn’s-like Enterocutaneous Fistula. Stem Cell Rev. Rep. 2020, 16, 1292–1304. [Google Scholar] [CrossRef]
  146. Li, Q.; Lian, Y.; Deng, Y.; Chen, J.; Wu, T.; Lai, X.; Zheng, B.; Qiu, C.; Peng, Y.; Li, W.; et al. mRNA-engineered mesenchymal stromal cells expressing CXCR2 enhances cell migration and improves recovery in IBD. Mol. Ther.-Nucleic Acids 2021, 26, 222–236. [Google Scholar] [CrossRef]
  147. Hansen, M.; Stahl, L.; Heider, A.; Hilger, N.; Sack, U.; Kirschner, A.; Cross, M.; Fricke, S. Reduction of Graft-versus-Host-Disease in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ. (NSG) Mice by Cotransplantation of Syngeneic Human Umbilical Cord-Derived Mesenchymal Stromal Cells: M. Transplant. Cell. Ther. 2021, 27, 658.e1–658.e10. [Google Scholar] [CrossRef] [PubMed]
  148. Augustine, S.; Cheng, W.; Avey, M.T.; Chan, M.L.; Lingappa, S.M.C.; Hutton, B.; Thébaud, B. Are all stem cells equal? Systematic review, evidence map, and meta-analyses of preclinical stem cell-based therapies for bronchopulmonary dysplasia. Stem Cells Transl. Med. 2020, 9, 158–168. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  149. Ee, M.T.; Thébaud, B. The Therapeutic Potential of Stem Cells for Bronchopulmonary Dysplasia: “It’s About Time” or “Not so Fast”? Curr. Pediatr. Rev. 2018, 14, 227–238. [Google Scholar] [CrossRef] [PubMed]
  150. Aslam, M.; Baveja, R.; Liang, O.D.; Fernandez-Gonzalez, A.; Lee, C.; Mitsialis, S.A.; Kourembanas, S. Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease. Am. J. Respir. Crit. Care Med. 2009, 180, 1122–1130. [Google Scholar] [CrossRef] [Green Version]
  151. Tropea, K.A.; Leder, E.; Aslam, M.; Lau, A.N.; Raiser, D.M.; Lee, J.H.; Balasubramaniam, V.; Fredenburgh, L.E.; Mitsialis, S.A.; Kourembanas, S.; et al. Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia. Am. J. Physiol.-Lung Cell. Mol. Physiol. 2012, 302, 829–837. [Google Scholar] [CrossRef]
  152. Hansmann, G.; Fernandez-Gonzalez, A.; Aslam, M.; Vitali, S.H.; Martin, T.; Alex Mitsialis, S.; Kourembanas, S. Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension. Pulm. Circ. 2012, 2, 170–181. [Google Scholar] [CrossRef] [Green Version]
  153. Zhang, H.; Fang, J.; Su, H.; Yang, M.; Lai, W.; Mai, Y.; Wu, Y. Bone marrow mesenchymal stem cells attenuate lung inflammation of hyperoxic newborn rats. Pediatr. Transplant. 2012, 16, 589–598. [Google Scholar] [CrossRef]
  154. Khemani, R.G.; Smith, L.; Lopez-Fernandez, Y.M.; Kwok, J.; Morzov, R.; Klein, M.J.; Yehya, N.; Willson, D.; Kneyber, M.C.J.; Lillie, J.; et al. Paediatric acute respiratory distress syndrome incidence and epidemiology (PARDIE): An international, observational study. Lancet Respir. Med. 2019, 7, 115–128. [Google Scholar] [CrossRef]
  155. Behnke, J.; Kremer, S.; Shahzad, T.; Chao, C.M.; Böttcher-Friebertshäuser, E.; Morty, R.E.; Bellusci, S.; Ehrhardt, H. MSC based therapies—new perspectives for the injured lung. J. Clin. Med. 2020, 9, 682. [Google Scholar] [CrossRef] [Green Version]
  156. Chen, J.; Luo, L.; Tian, R.; Yu, C. A review and update for registered clinical studies of stem cells for non-tumorous and non-hematological diseases. Regen. Ther. 2021, 18, 355–362. [Google Scholar] [CrossRef]
  157. Sondhi, D.; Peterson, D.A.; Edelstein, A.M.; del Fierro, K.; Hackett, N.R.; Crystal, R.G. Survival advantage of neonatal CNS gene transfer for late infantile neuronal ceroid lipofuscinosis. Exp. Neurol. 2008, 213, 18–27. [Google Scholar] [CrossRef] [Green Version]
  158. Ahmed, S.S.; Li, H.; Cao, C.; Sikoglu, E.M.; Denninger, A.R.; Su, Q.; Eaton, S.; Liso Navarro, A.A.; Xie, J.; Szucs, S.; et al. A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS gene therapy in Canavan mice. Mol. Ther. 2013, 21, 2136–2147. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  159. Foust, K.D.; Wang, X.; McGovern, V.L.; Braun, L.; Bevan, A.K.; Haidet, A.M.; Le, T.T.; Morales, P.R.; Rich, M.M.; Burghes, A.H.M.; et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat. Biotechnol. 2010, 28, 271–274. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  160. Cabrera-Salazar, M.A.; Roskelley, E.M.; Bu, J.; Hodges, B.L.; Yew, N.; Dodge, J.C.; Shihabuddin, L.S.; Sohar, I.; Sleat, D.E.; Scheule, R.K.; et al. Timing of therapeutic intervention determines functional and survival outcomes in a mouse model of late infantile batten disease. Mol. Ther. 2007, 15, 1782–1788. [Google Scholar] [CrossRef] [PubMed]
  161. Fu, H.; Cataldi, M.P.; Ware, T.A.; Zaraspe, K.; Meadows, A.S.; Murrey, D.A.; McCarty, D.M. Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery. Mol. Ther.-Methods Clin. Dev. 2016, 3, 16036. [Google Scholar] [CrossRef] [Green Version]
  162. Johnson, R.; Rafuse, M.; Selvakumar, P.P.; Tan, W. Effects of recipient age, heparin release and allogeneic bone marrow-derived stromal cells on vascular graft remodeling. Acta Biomater. 2021, 125, 172–182. [Google Scholar] [CrossRef]
  163. Themis, M.; Waddington, S.N.; Schmidt, M.; von Kalle, C.; Wang, Y.; Al-Allaf, F.; Gregory, L.G.; Nivsarkar, M.; Themis, M.; Holder, M.V.; et al. Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice. Mol. Ther. 2005, 12, 763–771. [Google Scholar] [CrossRef]
  164. Nowrouzi, A.; Cheung, W.T.; Li, T.; Zhang, X.; Arens, A.; Paruzynski, A.; Waddington, S.N.; Osejindu, E.; Reja, S.; von Kalle, C.; et al. The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis. Mol. Ther. 2013, 21, 324–337. [Google Scholar] [CrossRef] [Green Version]
  165. Riley, R.S.; Kashyap, M.V.; Billingsley, M.M.; White, B.; Alameh, M.G.; Bose, S.K.; Zoltick, P.W.; Li, H.; Zhang, R.; Cheng, A.Y.; et al. Ionizable lipid nanoparticles for in utero mRNA delivery. Sci. Adv. 2021, 7, eaba1028. [Google Scholar] [CrossRef]
  166. Borrell, V.; Yoshimura, Y.; Callaway, E.M. Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J. Neurosci. Methods 2005, 143, 151–158. [Google Scholar] [CrossRef]
  167. Joyeux, L.; Danzer, E.; Limberis, M.P.; Zoltick, P.W.; Radu, A.; Flake, A.W.; Davey, M.G. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice. Hum. Gene Ther. Methods 2014, 25, 197–205. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  168. Sabatino, D.E.; MacKenzie, T.C.; Peranteau, W.; Edmonson, S.; Campagnoli, C.; Liu, Y.L.; Flake, A.W.; High, K.A. Persistent expression of hF.IX after tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol. Ther. 2007, 15, 1677–1685. [Google Scholar] [CrossRef] [PubMed]
  169. Bose, S.K.; White, B.M.; Kashyap, M.V.; Dave, A.; De Bie, F.R.; Li, H.; Singh, K.; Menon, P.; Wang, T.; Teerdhala, S.; et al. In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease. Nat. Commun. 2021, 12, 4291. [Google Scholar] [CrossRef] [PubMed]
  170. Massaro, G.; Mattar, C.N.Z.; Wong, A.M.S.; Sirka, E.; Buckley, S.M.K.; Herbert, B.R.; Karlsson, S.; Perocheau, D.P.; Burke, D.; Heales, S.; et al. Fetal gene therapy for neurodegenerative disease of infants. Nat. Med. 2018, 24, 1317–1323. [Google Scholar] [CrossRef] [PubMed]
  171. Rossidis, A.C.; Stratigis, J.D.; Chadwick, A.C.; Hartman, H.A.; Ahn, N.J.; Li, H.; Singh, K.; Coons, B.E.; Li, L.; Lv, W.; et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 2018, 24, 1513–1518. [Google Scholar] [CrossRef]
  172. Chan, J.K.Y.Y.; Gil-Farina, I.; Johana, N.; Rosales, C.; Tan, Y.W.; Ceiler, J.; Mcintosh, J.; Ogden, B.; Waddington, S.N.; Schmidt, M.; et al. Therapeutic expression of human clotting factors IX and × following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques. FASEB J. 2019, 33, 3954–3967. [Google Scholar] [CrossRef] [Green Version]
  173. Alapati, D.; Zacharias, W.J.; Hartman, H.A.; Rossidis, A.C.; Stratigis, J.D.; Ahn, N.J.; Coons, B.; Zhou, S.; Li, H.; Singh, K.; et al. In utero gene editing for monogenic lung disease. Sci. Transl. Med. 2019, 11, eaav8375. [Google Scholar] [CrossRef] [Green Version]
  174. Dighe, N.M.; Tan, K.W.; Tan, L.G.; Shaw, S.S.W.; Buckley, S.M.K.; Sandikin, D.; Johana, N.; Tan, Y.W.; Biswas, A.; Choolani, M.; et al. A comparison of intrauterine hemopoietic cell transplantation and lentiviral gene transfer for the correction of severe β-thalassemia in a HbbTh3/+ murine model. Exp. Hematol. 2018, 62, 45–55. [Google Scholar] [CrossRef] [Green Version]
  175. Ricciardi, A.S.; Bahal, R.; Farrelly, J.S.; Quijano, E.; Bianchi, A.H.; Luks, V.L.; Putman, R.; López-Giráldez, F.; Coşkun, S.; Song, E.; et al. In utero nanoparticle delivery for site-specific genome editing. Nat. Commun. 2018, 9, 2481. [Google Scholar] [CrossRef] [Green Version]
  176. Kumar, P.; Gao, K.; Wang, C.; Pivetti, C.; Lankford, L.; Farmer, D.; Wang, A. In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A. Cell Transplant. 2018, 27, 130–139. [Google Scholar] [CrossRef]
  177. Hayashi, S.; Abdulmalik, O.; Peranteau, W.H.; Ashizuka, S.; Campagnoli, C.; Chen, Q.; Horiuchi, K.; Asakura, T.; Flake, A.W. Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy. Exp. Hematol. 2003, 31, 176–184. [Google Scholar] [CrossRef]
  178. Meza, N.W.; Alonso-Ferrero, M.E.; Navarro, S.; Quintana-Bustamante, O.; Valeri, A.; Garcia-Gomez, M.; Bueren, J.A.; Bautista, J.M.; Segovia, J.C. Rescue of pyruvate kinase deficiency in mice by gene therapy using the human isoenzyme. Mol. Ther. 2009, 17, 2000–2009. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  179. Loukogeorgakis, S.P.; Shangaris, P.; Bertin, E.; Franzin, C.; Piccoli, M.; Pozzobon, M.; Subramaniam, S.; Tedeschi, A.; Kim, A.G.; Li, H.; et al. In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment. Stem Cells 2019, 37, 1176–1188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  180. Shangaris, P.; Loukogeorgakis, S.P.; Blundell, M.P.; Petra, E.; Shaw, S.W.; Ramachandra, D.L.; Maghsoudlou, P.; Urbani, L.; Thrasher, A.J.; De Coppi, P.; et al. Long-Term Hematopoietic Engraftment of Congenic Amniotic Fluid Stem Cells After in Utero Intraperitoneal Transplantation to Immune Competent Mice. Stem Cells Dev. 2018, 27, 515–523. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  181. Hayashi, M.; Muramatsu, H.; Nakano, M.; Ito, H.; Inoie, M.; Tomizuka, Y.; Inoue, M.; Yoshimoto, S. Experience of using cultured epithelial autografts for the extensive burn wounds in eight patients. Ann. Plast. Surg. 2014, 73, 25–29. [Google Scholar] [CrossRef]
  182. Hoburg, A.; Löer, I.; Körsmeier, K.; Siebold, R.; Niemeyer, P.; Fickert, S.; Ruhnau, K. Matrix-Associated Autologous Chondrocyte Implantation Is an Effective Treatment at Midterm Follow-up in Adolescents and Young Adults. Orthop. J. Sport. Med. 2019, 7, 1–7. [Google Scholar] [CrossRef] [Green Version]
  183. Schmidt, C. Gintuit cell therapy approval signals shift at US regulator. Nat. Biotechnol. 2012, 30, 479. [Google Scholar] [CrossRef]
  184. Takaya, K.; Kato, T.; Ishii, T.; Sakai, S.; Okabe, K.; Aramaki-Hattori, N.; Asou, T.; Kishi, K. Clinical Analysis of Cultured Epidermal Autograft (JACE) Transplantation for Giant Congenital Melanocytic Nevus. Plast. Reconstr. Surg.-Glob. Open 2021, 9, e3380. [Google Scholar] [CrossRef]
  185. Eudy, M.; Eudy, C.L.; Roy, S. Apligraf as an Alternative to Skin Grafting in the Pediatric Population. Cureus 2021, 13, e16226. [Google Scholar] [CrossRef]
  186. Mavilio, F.; Pellegrini, G.; Ferrari, S.; Di Nunzio, F.; Di Iorio, E.; Recchia, A.; Maruggi, G.; Ferrari, G.; Provasi, E.; Bonini, C.; et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat. Med. 2006, 12, 1397–1402. [Google Scholar] [CrossRef]
  187. Hirsch, T.; Rothoeft, T.; Teig, N.; Bauer, J.W.; Pellegrini, G.; De Rosa, L.; Scaglione, D.; Reichelt, J.; Klausegger, A.; Kneisz, D.; et al. Regeneration of the entire human epidermis using transgenic stem cells. Nature 2017, 551, 327–332. [Google Scholar] [CrossRef] [PubMed]
  188. Di, W.L.; Lwin, S.M.; Petrova, A.; Bernadis, C.; Syed, F.; Farzaneh, F.; Moulding, D.; Martinez, A.E.; Sebire, N.J.; Rampling, D.; et al. Generation and Clinical Application of Gene-Modified Autologous Epidermal Sheets in Netherton Syndrome: Lessons Learned from a Phase 1 Trial. Hum. Gene Ther. 2019, 30, 1067–1078. [Google Scholar] [CrossRef] [PubMed]
  189. Siprashvili, Z.; Nguyen, N.T.; Gorell, E.S.; Loutit, K.; Khuu, P.; Furukawa, L.K.; Lorenz, H.P.; Leung, T.H.; Keene, D.R.; Rieger, K.E.; et al. Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients with Recessive Dystrophic Epidermolysis Bullosa. JAMA 2016, 316, 1808–1817. [Google Scholar] [CrossRef] [PubMed]
  190. European Medicines Agency. PRIME: Priority Medicines|European Medicines Agency. Available online: (accessed on 30 December 2021).
  191. Inacio, P. Amicus Discontinues Gene Therapy Program for CLN6 Batten Disease. Available online: (accessed on 28 February 2022).
  192. Pearson, A.D.J.; Rossig, C.; Lesa, G.; Diede, S.J.; Weiner, S.; Anderson, J.; Gray, J.; Geoerger, B.; Minard-Colin, V.; Marshall, L.V.; et al. ACCELERATE and European Medicines Agency. Paediatric Strategy Forum for medicinal product development of checkpoint inhibitors for use in combination therapy in paediatric patients. Eur. J. Cancer 2020, 127, 52–66. [Google Scholar] [CrossRef] [Green Version]
  193. Buckland, K.F.; Bobby Gaspar, H. Gene and cell therapy for children--new medicines, new challenges? Adv. Drug Deliv. Rev. 2014, 73, 162–169. [Google Scholar] [CrossRef] [Green Version]
  194. Marktel, S.; Scaramuzza, S.; Cicalese, M.P.; Giglio, F.; Galimberti, S.; Lidonnici, M.R.; Calbi, V.; Assanelli, A.; Bernardo, M.E.; Rossi, C.; et al. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia. Nat. Med. 2019, 25, 234–241. [Google Scholar] [CrossRef]
  195. DeWeerdt, S. Prenatal gene therapy offers the earliest possible cure. Nature 2018, 564, S6–S8. [Google Scholar] [CrossRef]
  196. Escolar, M.L.; Poe, M.D.; Provenzale, J.M.; Richards, K.C.; Allison, J.; Wood, S.; Wenger, D.A.; Pietryga, D.; Wall, D.; Champagne, M.; et al. Transplantation of Umbilical-Cord Blood in Babies with Infantile Krabbe’s Disease. N. Engl. J. Med. 2005, 352, 2069–2081. [Google Scholar] [CrossRef] [Green Version]
  197. Gray, S.J. Timing of Gene Therapy Interventions: The Earlier, the Better. Mol. Ther. 2016, 24, 1017–1018. [Google Scholar] [CrossRef] [Green Version]
  198. Garrison, L.P.; Jackson, T.; Paul, D.; Kenston, M. Value-based pricing for emerging gene therapies: The economic case for a higher cost-effectiveness threshold. J. Manag. Care Spec. Pharm. 2019, 25, 793–799. [Google Scholar] [CrossRef]
  199. Conti, R.; Gruber, J.; Ollendorf, D.; Neumann, P. Valuing Rare Pediatric Drugs: An Economics Perspective. SSRN Electron. J. 2021. NBER Working Paper No. w27978. [Google Scholar] [CrossRef]
  200. Bolous, N.S.; Chen, Y.; Wang, H.; Davidoff, A.M.; Devidas, M.; Jacobs, T.W.; Meagher, M.M.; Nathwani, A.C.; Neufeld, E.J.; Piras, B.A.; et al. The cost-effectiveness of gene therapy for severe hemophilia B: A microsimulation study from the United States perspective. Blood 2021, 138, 1677–1690. [Google Scholar] [CrossRef] [PubMed]
  201. Aiuti, A.; Roncarolo, M.G.; Naldini, L. Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: Paving the road for the next generation of advanced therapy medicinal products. EMBO Mol. Med. 2017, 9, 737–740. [Google Scholar] [CrossRef] [PubMed]
  202. Fumagalli, F.; Calbi, V.; Natali Sora, M.G.; Sessa, M.; Baldoli, C.; Rancoita, P.M.V.; Ciotti, F.; Sarzana, M.; Fraschini, M.; Zambon, A.A.; et al. Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: Long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access. Lancet 2022, 399, 372–383. [Google Scholar] [CrossRef]
  203. Matteini, F.; Mulaw, M.A.; Florian, M.C. Aging of the Hematopoietic Stem Cell Niche: New Tools to Answer an Old Question. Front. Immunol. 2021, 12, 738204. [Google Scholar] [CrossRef]
  204. Garcia, O.; Carraro, G.; Navarro, S.; Bertoncello, I.; McQualter, J.; Driscoll, B.; Jesudason, E.; Warburton, D. Cell-based therapies for lung disease. Br. Med. Bull. 2012, 101, 147–161. [Google Scholar] [CrossRef] [Green Version]
  205. Alofisel|European Medicines Agency. Available online: (accessed on 30 December 2021).
  206. Kuçi, Z.; Bönig, H.; Kreyenberg, H.; Bunos, M.; Jauch, A.; Janssen, J.W.G.; Škifić, M.; Michel, K.; Eising, B.; Lucchini, G.; et al. Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: A multicenter survey. Haematologica 2016, 101, 985–994. [Google Scholar] [CrossRef]
  207. Kurtzberg, J.; Prockop, S.; Teira, P.; Bittencourt, H.; Lewis, V.; Chan, K.W.; Horn, B.; Yu, L.; Talano, J.A.; Nemecek, E.; et al. Allogeneic human mesenchymal stem cell therapy (Remestemcel-L, Prochymal) as a rescue agent for severe refractory acute graft-versus-host disease in pediatric patients. Biol. Blood Marrow Transplant. 2014, 20, 229–235. [Google Scholar] [CrossRef] [Green Version]
  208. Lucchini, G.; Introna, M.; Dander, E.; Rovelli, A.; Balduzzi, A.; Bonanomi, S.; Salvadè, A.; Capelli, C.; Belotti, D.; Gaipa, G.; et al. Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population. Biol. Blood Marrow Transplant. 2010, 16, 1293–1301. [Google Scholar] [CrossRef] [Green Version]
  209. Prasad, V.K.; Lucas, K.G.; Kleiner, G.I.; Talano, J.A.M.; Jacobsohn, D.; Broadwater, G.; Monroy, R.; Kurtzberg, J. Efficacy and Safety of Ex Vivo Cultured Adult Human Mesenchymal Stem Cells (ProchymalTM) in Pediatric Patients with Severe Refractory Acute Graft-Versus-Host Disease in a Compassionate Use Study. Biol. Blood Marrow Transplant. 2011, 17, 534–541. [Google Scholar] [CrossRef] [Green Version]
  210. Zilberberg, J.; Friedman, T.M.; Korngold, R.; Szabolcs, P.; Visani, G.; Locatelli, F.; Kleiner, G.; Nishida, T.; Onizuka, M.; Inamoto, Y.; et al. Treatment Of Steroid-Refractory Acute GVHD with Mesenchymal Stem Cells Improves Outcomes In Pediatric Patients; Results Of The Pediatric Subset In A Phase III Randomized, Placebo-Controlled Study. Biol. Blood Marrow Transplant. 2010, 16, S298. [Google Scholar] [CrossRef] [Green Version]
  211. MacMillan, M.L.; Blazar, B.R.; DeFor, T.E.; Wagner, J.E. Transplantation of ex-vivo culture-expanded parental haploidentical mesenchymal stem cells to promote engraftment in pediatric recipients of unrelated donor umbilical cord blood: Results of a phase I-II clinical trial. Bone Marrow Transplant. 2009, 43, 447–454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  212. Lee, S.H.; Lee, M.W.; Yoo, K.H.; Kim, D.S.; Son, M.H.; Sung, K.W.; Cheuh, H.; Choi, S.J.; Oh, W.; Yang, Y.S.; et al. Co-transplantation of third-party umbilical cord blood-derived MSCs promotes engraftment in children undergoing unrelated umbilical cord blood transplantation. Bone Marrow Transplant. 2013, 48, 1040–1045. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  213. Bernardo, M.E.; Ball, L.M.; Cometa, A.M.; Roelofs, H.; Zecca, M.; Avanzini, M.A.; Bertaina, A.; Vinti, L.; Lankester, A.; MacCario, R.; et al. Co-infusion of ex vivo-expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation. Bone Marrow Transplant. 2011, 46, 200–207. [Google Scholar] [CrossRef] [Green Version]
  214. Voynow, J.A. “New” bronchopulmonary dysplasia and chronic lung disease. Paediatr. Respir. Rev. 2017, 24, 17–18. [Google Scholar] [CrossRef]
  215. Möbius, M.A.; Thébaud, B. Cell Therapy for Bronchopulmonary Dysplasia: Promises and Perils. Paediatr. Respir. Rev. 2016, 20, 33–41. [Google Scholar] [CrossRef]
  216. Fujinaga, H.; Baker, C.D.; Ryan, S.L.; Markham, N.E.; Seedorf, G.J.; Balasubramaniam, V.; Abman, S.H. Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants. Am. J. Physiol.-Lung Cell. Mol. Physiol. 2009, 297, 1160–1169. [Google Scholar] [CrossRef] [Green Version]
  217. Chang, Y.S.; Ahn, S.Y.; Yoo, H.S.; Sung, S.I.; Choi, S.J.; Oh, W.I.; Park, W.S. Mesenchymal Stem Cells for Bronchopulmonary Dysplasia: Phase 1 Dose-Escalation Clinical Trial. J. Pediatr. 2014, 164, 966–972.e6. [Google Scholar] [CrossRef]
  218. Ahn, S.Y.; Chang, Y.S.; Kim, J.H.; Sung, S.I.; Park, W.S. Two-Year Follow-Up Outcomes of Premature Infants Enrolled in the Phase I Trial of Mesenchymal Stem Cells Transplantation for Bronchopulmonary Dysplasia. J. Pediatr. 2017, 185, 49–54.e2. [Google Scholar] [CrossRef] [Green Version]
  219. Medipost Co Ltd. Long-Term Safety and Efficacy Follow-Up Study of PNEUMOSTEM® in Patients Who Completed PNEUMOSTEM® Phase-I Study. Available online: (accessed on 8 December 2021).
  220. Powell, S.B.; Silvestri, J.M. Safety of Intratracheal Administration of Human Umbilical Cord Blood Derived Mesenchymal Stromal Cells in Extremely Low Birth Weight Preterm Infants. J. Pediatr. 2019, 210, 209–213.e2. [Google Scholar] [CrossRef]
  221. Jouvet, P.; Thomas, N.J.; Willson, D.F.; Erickson, S.; Khemani, R.; Smith, L.; Zimmerman, J.; Dahmer, M.; Flori, H.; Quasney, M.; et al. Pediatric Acute Respiratory Distress Syndrome: Consensus Recommendations from the Pediatric Acute Lung Injury Consensus Conference. Pediatr. Crit. Care Med. 2015, 16, 428–439. [Google Scholar] [CrossRef] [Green Version]
  222. Heidemann, S.M.; Nair, A.; Bulut, Y.; Sapru, A. Pathophysiology and Management of Acute Respiratory Distress Syndrome in Children. Pediatr. Clin. N. Am. 2017, 64, 1017–1037. [Google Scholar] [CrossRef] [PubMed]
  223. Thompson, B.T.; Chambers, R.C.; Liu, K.D. Acute Respiratory Distress Syndrome. N. Engl. J. Med. 2017, 377, 562–572. [Google Scholar] [CrossRef] [PubMed]
  224. Wilson, J.G.; Liu, K.D.; Zhuo, H.; Caballero, L.; McMillan, M.; Fang, X.; Cosgrove, K.; Vojnik, R.; Calfee, C.S.; Lee, J.W.; et al. Mesenchymal stem (stromal) cells for treatment of ARDS: A phase 1 clinical trial. Lancet Respir. Med. 2015, 3, 24–32. [Google Scholar] [CrossRef] [Green Version]
  225. Weiss, D.J. Cell-based therapies for acute respiratory distress syndrome. Lancet Respir. Med. 2019, 7, 105–106. [Google Scholar] [CrossRef]
  226. Matthay, M.A.; Calfee, C.S.; Zhuo, H.; Thompson, B.T.; Wilson, J.G.; Levitt, J.E.; Rogers, A.J.; Gotts, J.E.; Wiener-Kronish, J.P.; Bajwa, E.K.; et al. Treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (START study): A randomised phase 2a safety trial. Lancet Respir. Med. 2019, 7, 154–162. [Google Scholar] [CrossRef]
  227. Ahmed, S.; Flinn, I.W.; Mei, M.; Riedell, P.A.; Armand, P.; Grover, N.S.; Engert, A.; Lapteva, N.; Nadler, P.I.; Myo, A.; et al. Safety and Efficacy Profile of Autologous CD30.CAR-T-Cell Therapy in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma (CHARIOT Trial). Blood 2021, 138, 3847–3850. [Google Scholar] [CrossRef]
  228. Tessa Therapeutics. Phase 2 Study Evaluating Autologous CD30.CAR-T Cells in Patients with Relapsed/Refractory Hodgkin Lymphoma (CHARIOT). Available online: (accessed on 21 March 2022).
  229. Nichols, H.; Eides, R. Vertex and CRISPR Therapeutics Present New Data in 22 Patients with Greater than 3 Months Follow-Up Post-Treatment with Investigational CRISPR/Cas9 Gene-Editing Therapy, CTX001TM. Available online: (accessed on 21 March 2022).
  230. CRISPRTX. A Safety and Efficacy Study Evaluating CTX001 in Subjects with Severe Sickle Cell Disease. Available online: (accessed on 21 March 2022).
  231. Aiuiti, A and IRCCS San Raffaele. Gene Therapy for Transfusion Dependent Beta-thalassemia (TIGET-BTHAL). Available online: (accessed on 27 February 2022).
  232. Orchard Therapeutics. Long-Term Follow-Up of Subjects Treated with OTL-300 for Transfusion-Dependent Beta-Thalassemia Study (TIGET-BTHAL). Available online: (accessed on 21 March 2022).
  233. Kohn, D.B.; Booth, C.; Sevilla, J.; Rao, G.R.; Almarza, E.; Terrazas, D.; Nicoletti, E.; Fernandes, A.; Kuo, C.; de Oliveira, S.; et al. A Phase 1/2 Study of Lentiviral-Mediated Ex-Vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I): Interim Results. Blood 2021, 138, 2932. [Google Scholar] [CrossRef]
  234. Kohn, D.B.; Rao, G.R.; Almarza, E.; Terrazas, D.; Nicoletti, E.; Fernandes, A.; Kuo, C.; De Oliveira, S.N.; Moore, T.B.; Law, K.M.; et al. A Phase 1/2 Study of Lentiviral-Mediated Ex-Vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I): Results from Phase 1. Blood 2020, 136, 15. [Google Scholar] [CrossRef]
  235. Rocket Pharmaceuticals Inc. A Clinical Trial to Evaluate the Safety and Efficacy of RP-L201 in Subjects with Leukocyte Adhesion Deficiency-I. Available online: (accessed on 21 March 2022).
  236. Aiuiti, A and IRCCS San Raffaele. Gene Therapy with Modified Autologous Hematopoietic Stem Cells for the Treatment of Patients with Mucopolysaccharidosis Type I, Hurler Variant (TigetT10_MPSIH). Available online: (accessed on 21 March 2022).
  237. Gentner, B.; Tucci, F.; Galimberti, S.; Fumagalli, F.; De Pellegrin, M.; Silvani, P.; Camesasca, C.; Pontesilli, S.; Darin, S.; Ciotti, F.; et al. Hematopoietic Stem-and Progenitor-Cell Gene Therapy for Hurler Syndrome. N. Engl. J. Med. 2021, 385, 1929–1940. [Google Scholar] [CrossRef]
  238. National Institutes of Health Clinical Center (CC) and National Institute of Allergy and Infectious Diseases (NIAID). Lentiviral Gene Transfer for Treatment of Children Older than 2 Years of Age with X-Linked Severe Combined Immunodeficiency (LVXSCID-OC). Available online: (accessed on 21 March 2022).
  239. De Ravin, S.S.; Anaya O’Brien, S.; Kwatemaa, N.; Theobald, N.; Liu, S.; Lee, J.; Kardava, L.; Liu, T.; Goldman, F.; Moir, S.; et al. Enhanced Transduction Lentivector Gene Therapy for Treatment of Older Patients with X-Linked Severe Combined Immunodeficiency. Blood 2019, 134 (Suppl. 1), 608. [Google Scholar] [CrossRef]
  240. Mamcarz, E.; Zhou, S.; Lockey, T.; Abdelsamed, H.; Cross, S.J.; Kang, G.; Ma, Z.; Condori, J.; Dowdy, J.; Triplett, B.; et al. Lentiviral gene therapy combined with low-dose busulfan in infants with SCID-X1. N. Engl. J. Med. 2019, 380, 1525–1534. [Google Scholar] [CrossRef] [PubMed]
  241. St. Jude Children′s Research Hospital. Gene Transfer for X-Linked Severe Combined Immunodeficiency in Newly Diagnosed Infants (LVXSCID-ND). Available online: (accessed on 21 March 2022).
  242. ExCellThera Inc. US Phase I Study of ECT-001-CB in Patients with Sickle-Cell Disease. Available online: (accessed on 21 March 2022).
  243. ExCellThera Inc. US Study of ECT-001-CB in Pediatric and Young Adult Patients with High-Risk Myeloid Malignancies. Available online: (accessed on 21 March 2022).
  244. Rocket Pharmaceuticals Inc. A Clinical Trial to Evaluate the Safety of RP-L102 in Pediatric Subjects with Fanconi Anemia Subtype A. Available online: (accessed on 21 March 2022).
  245. Czechowicz, A.; Roncarolo, M.G.; Beard, B.C.; Law, K.; Nicoletti, E.; Río, P.; Bueren, J.A.; Schwartz, J.D.; Soni, S. Changing the Natural History of Fanconi Anemia Complementation Group-A with Gene Therapy: Early Results of U.S. Phase I Study of Lentiviral-Mediated Ex-VivoFANCA Gene Insertion in Human Stem and Progenitor Cells. Blood 2019, 134, 3350. [Google Scholar] [CrossRef]
  246. Rocket Pharmaceuticals Inc. Gene Therapy for Fanconi Anemia, Complementation Group A. Available online: (accessed on 21 March 2022).
  247. Rocket Pharmaceuticals Inc. Lentiviral-Mediated Gene Therapy for Pediatric Patients with Fanconi Anemia Subtype A. Available online: (accessed on 21 March 2022).
  248. bluebird bio. Longterm Follow-Up of Subjects with Hemoglobinopathies Treated with Ex Vivo Gene Therapy. Available online: (accessed on 21 March 2022).
  249. AlloVir. Study of Viralym-M (ALVR105) for Multi-Virus Prevention in Patients Post-Allogeneic Hematopoietic Cell Transplant. Available online: (accessed on 21 March 2022).
  250. Dadwal, S.S.; Shuster, M.; Myers, G.D.; Boundy, K.; Warren, M.; Stoner, E.; Truong, T.; Hill, J.A. Posoleucel (ALVR105), an Off-the-Shelf, Multivirus-Specific T-Cell Therapy, for the Prevention of Viral Infections Post-HCT: Results from an Open-Label Cohort of a Phase 2 Trial. Blood 2021, 138, 1760. [Google Scholar] [CrossRef]
  251. AlloVir. Study to Evaluate Viralym-M (ALVR105) for the Treatment of Virus-Associated Hemorrhagic Cystitis (HC). Available online: (accessed on 21 March 2022).
  252. Elbashir, S.M.; Martinez, J.; Patkaniowska, A.; Lendeckel, W.; Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 2001, 20, 6877–6888. [Google Scholar] [CrossRef] [Green Version]
  253. Ahn, S.Y.; Chang, Y.S.; Lee, M.H.; Sung, S.I.; Lee, B.S.; Kim, K.S.; Kim, A.R.; Park, W.S. Stem cells for bronchopulmonary dysplasia in preterm infants: A randomized controlled phase II trial. Stem Cells Transl. Med. 2021, 10, 1129–1137. [Google Scholar] [CrossRef]
  254. Medipost Co Ltd. Follow-Up Safety and Efficacy Evaluation on Subjects Who Completed PNEUMOSTEM® Phase-II Clinical Trial. Available online: (accessed on 8 December 2021).
  255. Lee Farmer, D.; University of California, Davis. Cellular Therapy for In Utero Repair of Myelomeningocele—The CuRe Trial. Available online: (accessed on 11 December 2021).
  256. Götherström, C and Karolinska Institutet. Boost Brittle Bones before Birth (BOOSTB4). Available online: (accessed on 11 December 2021).
  257. Schneider, H.; Faschingbauer, F.; Schuepbach-Mallepell, S.; Körber, I.; Wohlfart, S.; Dick, A.; Wahlbuhl, M.; Kowalczyk-Quintas, C.; Vigolo, M.; Kirby, N.; et al. Prenatal Correction of X-Linked Hypohidrotic Ectodermal Dysplasia. N. Engl. J. Med. 2018, 378, 1604–1610. [Google Scholar] [CrossRef]
  258. Kreger, E.M.; Singer, S.T.; Witt, R.G.; Sweeters, N.; Lianoglou, B.; Lal, A.; Mackenzie, T.C.; Vichinsky, E. Favorable outcomes after in utero transfusion in fetuses with alpha thalassemia major: A case series and review of the literature. Prenat. Diagn. 2016, 36, 1242–1249. [Google Scholar] [CrossRef]
  259. Mackenzie, T.; University of California, San Francisco. In Utero Hematopoietic Stem Cell Transplantation for Alpha-Thalassemia Major (ATM). Available online: (accessed on 11 December 2021).
  260. MacKenzie, T.C.; Frascoli, M.; Sper, R.; Lianoglou, B.R.; Gonzalez Velez, J.; Dvorak, C.C.; Kharbanda, S.; Vichinsky, E. In Utero Stem Cell Transplantation in Patients with Alpha Thalassemia Major: Interim Results of a Phase 1 Clinical Trial. Blood 2020, 136 (Suppl. 1), 1. [Google Scholar] [CrossRef]
  261. Dimitri, P.; Pignataro, V.; Lupo, M.; Bonifazi, D.; Henke, M.; Musazzi, U.M.; Ernst, F.; Minghetti, P.; Redaelli, D.F.; Antimisiaris, S.G.; et al. Medical device development for children and young people—reviewing the challenges and opportunities. Pharmaceutics 2021, 13, 2178. [Google Scholar] [CrossRef]
  262. Fesnak, A.; O’Doherty, U. Clinical development and manufacture of chimeric antigen receptor T cells and the role of leukapheresis. Eur. Oncol. Haematol. 2017, 13, 28–34. [Google Scholar] [CrossRef] [Green Version]
  263. Cattoglio, C.; Facchini, G.; Sartori, D.; Antonelli, A.; Miccio, A.; Cassani, B.; Schmidt, M.; Von Kalle, C.; Howe, S.; Thrasher, A.J.; et al. Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood 2007, 110, 1770–1778. [Google Scholar] [CrossRef] [PubMed]
  264. Uchida, N.; Sutton, R.E.; Friera, A.M.; He, D.; Reitsma, M.J.; Chang, W.C.; Veres, G.; Scollay, R.; Weissman, I.L. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 1998, 95, 11939–11944. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  265. Lu, H.; Zhao, X.; Li, Z.; Hu, Y.; Wang, H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front. Oncol. 2021, 11, 720501. [Google Scholar] [CrossRef] [PubMed]
  266. Tonn, T.; Becker, S.; Esser, R.; Schwabe, D.; Seifried, E. Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J. Hematother. Stem Cell Res. 2001, 10, 535–544. [Google Scholar] [CrossRef]
  267. Bjordahl, R.; Gaidarova, S.; Goodridge, J.P.; Mahmood, S.; Bonello, G.; Robinson, M.; Ruller, C.; Pribadi, M.; Lee, T.; Abujarour, R.; et al. FT576: A Novel Multiplexed Engineered Off-the-Shelf Natural Killer Cell Immunotherapy for the Dual-Targeting of CD38 and Bcma for the Treatment of Multiple Myeloma. Blood 2019, 134, 3214. [Google Scholar] [CrossRef]
  268. Goodridge, J.P.; Mahmood, S.; Zhu, H.; Gaidarova, S.; Blum, R.; Bjordahl, R.; Cichocki, F.; Chu, H.; Bonello, G.; Lee, T.; et al. FT596: Translation of First-of-Kind Multi-Antigen Targeted Off-the-Shelf CAR-NK Cell with Engineered Persistence for the Treatment of B Cell Malignancies. Blood 2019, 134, 301. [Google Scholar] [CrossRef]
  269. Haspel, R.; Miller, K. Hematopoietic Stem Cells: Source Matters. Curr. Stem Cell Res. Ther. 2008, 3, 229–236. [Google Scholar] [CrossRef]
  270. Styczynski, J.; Balduzzi, A.; Gil, L.; Labopin, M.; Hamladji, R.M.; Marktel, S.; Yesilipek, M.A.; Fagioli, F.; Ehlert, K.; Matulova, M.; et al. Risk of complications during hematopoietic stem cell collection in pediatric sibling donors: A prospective European Group for Blood and Marrow Transplantation Pediatric Diseases Working Party study. Blood 2012, 119, 2935–2942. [Google Scholar] [CrossRef]
  271. Drabko, K. Autologous hematopoietic stem cell transplantation (auto-HSCT) in children in Poland: 2021 indications and practice. Acta Haematol. Pol. 2021, 52, 234–236. [Google Scholar] [CrossRef]
  272. Ohara, Y.; Ohto, H.; Tasaki, T.; Sano, H.; Mochizuki, K.; Akaihata, M.; Kobayashi, S.; Waragai, T.; Ito, M.; Hosoya, M.; et al. Comprehensive technical and patient-care optimization in the management of pediatric apheresis for peripheral blood stem cell harvesting. Transfus. Apher. Sci. 2016, 55, 338–343. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  273. Karakukcu, M.; Unal, E. Stem cell mobilization and collection from pediatric patients and healthy children. Transfus. Apher. Sci. 2015, 53, 17–22. [Google Scholar] [CrossRef] [PubMed]
  274. Lipton, J.M. Peripheral blood as a stem cell source for hematopoietic cell transplantation in children: Is the effort in vein? Pediatr. Transplant. 2003, 7, 65–70. [Google Scholar] [CrossRef] [PubMed]
  275. DiPersio, J.F.; Karpova, D.; Rettig, M.P. Mobilized peripheral blood: An updated perspective. F1000Research 2019, 8, 2125. [Google Scholar] [CrossRef] [Green Version]
  276. Luzzi, J.R.; Borba, C.C.; Miyaji, S.C.; Brito, C.A.; Navarro-Xavier, R.; Dinardo, C.L. Reduced volume of red blood cell priming is safe for pediatric patients undergoing therapeutic plasma exchange. Transfus. Apher. Sci. 2021, 60, 103005. [Google Scholar] [CrossRef]
  277. Luo, C.; Wang, L.; Wu, G.; Huang, X.; Zhang, Y.; Ma, Y.; Xie, M.; Sun, Y.; Huang, Y.; Huang, Z.; et al. Comparison of the efficacy of hematopoietic stem cell mobilization regimens: A systematic review and network meta-analysis of preclinical studies. Stem Cell Res. Ther. 2021, 12, 310. [Google Scholar] [CrossRef]
  278. Tajer, P.; Pike-Overzet, K.; Arias, S.; Havenga, M.; Staal, F. Ex Vivo Expansion of Hematopoietic Stem Cells for Therapeutic Purposes: Lessons from Development and the Niche. Cells 2019, 8, 169. [Google Scholar] [CrossRef] [Green Version]
  279. Baldwin, K.; Urbinati, F.; Romero, Z.; Campo-Fernandez, B.; Kaufman, M.L.; Cooper, A.R.; Masiuk, K.; Hollis, R.P.; Kohn, D.B. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy. Stem Cells 2015, 33, 1532–1542. [Google Scholar] [CrossRef] [Green Version]
  280. Jiang, W.; Xu, J. Immune modulation by mesenchymal stem cells. Cell Prolif. 2020, 53, e12712. [Google Scholar] [CrossRef]
  281. Ullah, I.; Subbarao, R.B.; Rho, G.J. Human mesenchymal stem cells-Current trends and future prospective. Biosci. Rep. 2015, 35, e00191. [Google Scholar] [CrossRef]
  282. Nehlin, J.O.; Jafari, A.; Tencerova, M.; Kassem, M. Aging and lineage allocation changes of bone marrow skeletal (stromal)stem cells. Bone 2019, 123, 265–273. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  283. Seo, Y.; Shin, T.H.; Kim, H.S. Current strategies to enhance adipose stem cell function: An update. Int. J. Mol. Sci. 2019, 20, 3827. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  284. Kuca-Warnawin, E.; Skalska, U.; Janicka, I.; Musiałowicz, U.; Bonek, K.; Głuszko, P.; Szczęsny, P.; Olesińska, M.; Kontny, E. The Phenotype and Secretory Activity of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Patients with Rheumatic Diseases. Cells 2019, 8, 1659. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  285. Ding, D.C.; Chang, Y.H.; Shyu, W.C.; Lin, S.Z. Human umbilical cord mesenchymal stem cells: A new era for stem cell therapy. Cell Transplant. 2015, 24, 339–347. [Google Scholar] [CrossRef] [PubMed]
  286. Goldman, S.A.; Schanz, S.; Windrem, M.S. Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol. Genet 2008, 17, R76–R83. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  287. Peng, Y.; Tang, L.; Zhou, Y. Subretinal Injection: A Review on the Novel Route of Therapeutic Delivery for Vitreoretinal Diseases. Ophthalmic Res. 2017, 58, 217–226. [Google Scholar] [CrossRef] [PubMed]
  288. Ramachandra, D.L.; Shaw, S.S.W.; Shangaris, P.; Loukogeorgakis, S.; Guillot, P.V.; De Coppi, P.; David, A.L. In utero therapy for congenital disorders using amniotic fluid stem cells. Front. Pharmacol. 2014, 5, 270. [Google Scholar] [CrossRef] [Green Version]
  289. Biological, K. Non Viral Vectors in Gene Therapy-An Overview. J. Clin. Diagn. Res. 2015, 9, GE01–GE06. [Google Scholar] [CrossRef]
  290. Boulaiz, H.; Marchal, J.A.; Prados, J.; Melguizo, C.; Aránega, A. Non-viral and viral vectors for gene therapy. Cell. Mol. Biol. 2005, 51, 3–22. [Google Scholar]
  291. Richter, M.; Stone, D.; Miao, C.; Humbert, O.; Kiem, H.P.; Papayannopoulou, T.; Lieber, A. In Vivo Hematopoietic Stem Cell Transduction. Hematol. Oncol. Clin. N. Am. 2017, 31, 771–785. [Google Scholar] [CrossRef]
  292. Murai, N.; Ohtaki, H.; Watanabe, J.; Xu, Z.; Sasaki, S.; Yagura, K.; Shioda, S.; Nagasaka, S.; Honda, K.; Izumizaki, M. Intrapancreatic injection of human bone marrow-derived mesenchymal stem/stromal cells alleviates hyperglycemia and modulates the macrophage state in streptozotocin-induced type 1 diabetic mice. PLoS ONE 2017, 12, e0186637. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  293. Li, C.; Georgakopoulou, A.; Mishra, A.; Gil, S.; Hawkins, R.D.; Yannaki, E.; Lieber, A. In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice. Blood Adv. 2021, 5, 1122–1135. [Google Scholar] [CrossRef] [PubMed]
  294. Schaefer, S.; Lange, S.; Werner, J.; Machka, C.; Neumann, K.; Knuebel, G.; Vogel, H.; Lindner, I.; Glass, Ä.; Escobar, H.M.; et al. Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model. Transplant. Cell. Ther. 2021, 28, 70.e1–70.e5. [Google Scholar] [CrossRef] [PubMed]
  295. Greber, U.F.; Gomez-Gonzalez, A. Adenovirus-a blueprint for gene delivery. Curr. Opin. Virol. 2021, 48, 49–56. [Google Scholar] [CrossRef]
  296. Chen, W.; Yao, S.; Wan, J.; Tian, Y.; Huang, L.; Wang, S.; Akter, F.; Wu, Y.; Yao, Y.; Zhang, X. BBB-crossing adeno-associated virus vector: An excellent gene delivery tool for CNS disease treatment. J. Control. Release 2021, 333, 129–138. [Google Scholar] [CrossRef]
  297. Jacobs, L.; De Smidt, E.; Geukens, N.; Declerck, P.; Hollevoet, K. Electroporation outperforms in vivo-jetPEI for intratumoral DNA-based reporter gene transfer. Sci. Rep. 2020, 10, 19532. [Google Scholar] [CrossRef]
  298. Kerstan, A.; Niebergall-Roth, E.; Esterlechner, J.; Schröder, H.M.; Gasser, M.; Waaga-Gasser, A.M.; Goebeler, M.; Rak, K.; Schrüfer, P.; Endres, S.; et al. Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: Process validation and first in-human data. Cytotherapy 2021, 23, 165–175. [Google Scholar] [CrossRef]
  299. Weiss, R.; Gerdes, W.; Berthold, R.; Sack, U.; Koehl, U.; Hauschildt, S.; Grahnert, A. Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells. Cells 2021, 10, 2824. [Google Scholar] [CrossRef]
  300. Han, L.; Zhou, J.; Li, L.; Zhou, K.; Zhao, L.; Zhu, X.; Yin, Q.; Li, Y.; You, H.; Zhang, J.; et al. Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies. Cancer Biol. Med. 2021, 18, 1066–1079. [Google Scholar] [CrossRef]
  301. Patsali, P.; Turchiano, G.; Papasavva, P.; Romito, M.; Loucari, C.C.; Stephanou, C.; Christou, S.; Sitarou, M.; Mussolino, C.; Cornu, T.I.; et al. Correction of IVS I-110(G>A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells. Haematologica 2019, 104, e497–e501. [Google Scholar] [CrossRef] [Green Version]
  302. Stephanou, C.; Papasavva, P.; Zachariou, M.; Patsali, P.; Epitropou, M.; Ladas, P.; Al-Abdulla, R.; Christou, S.; Antoniou, M.N.; Lederer, C.W.; et al. Suitability of small diagnostic peripheral-blood samples for cell-therapy studies. Cytotherapy 2017, 19, 311–326. [Google Scholar] [CrossRef] [PubMed]
  303. Robbins, G.M.; Wang, M.; Pomeroy, E.J.; Moriarity, B.S. Nonviral genome engineering of natural killer cells. Stem Cell Res. Ther. 2021, 12, 350. [Google Scholar] [CrossRef] [PubMed]
  304. Kim, J.Y.; Choi, J.H.; Kim, S.H.; Park, H.; Lee, D.; Kim, G.J. Efficacy of Gene Modification in Placenta-Derived Mesenchymal Stem Cells Based on Nonviral Electroporation. Int. J. Stem Cells 2021, 14, 112–118. [Google Scholar] [CrossRef] [PubMed]
  305. Holstein, M.; Mesa-Nuñez, C.; Miskey, C.; Almarza, E.; Poletti, V.; Schmeer, M.; Grueso, E.; Ordóñez Flores, J.C.; Kobelt, D.; Walther, W.; et al. Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors. Mol. Ther. 2018, 26, 1137–1153. [Google Scholar] [CrossRef] [Green Version]
  306. Lattanzi, A.; Meneghini, V.; Pavani, G.; Amor, F.; Ramadier, S.; Felix, T.; Antoniani, C.; Masson, C.; Alibeu, O.; Lee, C.; et al. Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements. Mol. Ther. 2019, 27, 137–150. [Google Scholar] [CrossRef] [Green Version]
  307. Russkamp, N.F.; Myburgh, R.; Kiefer, J.D.; Neri, D.; Manz, M.G. Anti-CD117 immunotherapy to eliminate hematopoietic and leukemia stem cells. Exp. Hematol. 2021, 95, 31–45. [Google Scholar] [CrossRef]
  308. Mangeot, P.E.; Risson, V.; Fusil, F.; Marnef, A.; Laurent, E.; Blin, J.; Mournetas, V.; Massouridès, E.; Sohier, T.J.M.; Corbin, A.; et al. Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Nat. Commun. 2019, 10, 45. [Google Scholar] [CrossRef] [Green Version]
  309. Li, C.; Psatha, N.; Wang, H.; Singh, M.; Samal, H.B.; Zhang, W.; Ehrhardt, A.; Izsvák, Z.; Papayannopoulou, T.; Lieber, A. Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies. Mol. Ther.-Methods Clin. Dev. 2018, 9, 142–152. [Google Scholar] [CrossRef] [Green Version]
  310. Banskota, S.; Raguram, A.; Suh, S.; Du, S.W.; Davis, J.R.; Choi, E.H.; Wang, X.; Nielsen, S.C.; Newby, G.A.; Randolph, P.B.; et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 2022, 185, 250–265. [Google Scholar] [CrossRef]
  311. Zu, H.; Gao, D. Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects. AAPS J. 2021, 23, 78. [Google Scholar] [CrossRef]
  312. Mangeot, P.E.; Guiguettaz, L.; Sohier, T.J.M.; Ricci, E.P. Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles (“Nanoblades”). J. Vis. Exp. 2021, 169, e62245. [Google Scholar] [CrossRef] [PubMed]
  313. He, X.; Urip, B.A.; Zhang, Z.; Ngan, C.C.; Feng, B. Evolving AAV-delivered therapeutics towards ultimate cures. J. Mol. Med. 2021, 99, 593–617. [Google Scholar] [CrossRef] [PubMed]
  314. Definition-Nanomaterials-Environment-European Commission. Available online: (accessed on 13 January 2021).
  315. Mitchell, M.J.; Billingsley, M.M.; Haley, R.M.; Wechsler, M.E.; Peppas, N.A.; Langer, R. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 2021, 20, 101–124. [Google Scholar] [CrossRef]
  316. DeLong, R.K.; Reynolds, C.M.; Malcolm, Y.; Schaeffer, A.; Severs, T.; Wanekaya, A. Functionalized gold nanoparticles for the binding, stabilization, and delivery of therapeutic DNA, RNA, and other biological macromolecules. Nanotechnol. Sci. Appl. 2010, 3, 53–63. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  317. Rai, R.; Alwani, S.; Badea, I. Polymeric nanoparticles in gene therapy: New avenues of design and optimization for delivery applications. Polymers 2019, 11, 745. [Google Scholar] [CrossRef] [Green Version]
  318. Tetro, N.; Moushaev, S.; Rubinchik-Stern, M.; Eyal, S. The Placental Barrier: The Gate and the Fate in Drug Distribution. Pharm. Res. 2018, 35, 71. [Google Scholar] [CrossRef]
  319. Muoth, C.; Aengenheister, L.; Kucki, M.; Wick, P.; Buerki-Thurnherr, T. Nanoparticle transport across the placental barrier: Pushing the field forward. Nanomedicine 2016, 11, 941–957. [Google Scholar] [CrossRef]
  320. Cruz, L.J.; van Dijk, T.; Vepris, O.; Li, T.M.W.Y.; Schomann, T.; Baldazzi, F.; Kurita, R.; Nakamura, Y.; Grosveld, F.; Philipsen, S.; et al. PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells. Biomaterials 2021, 268, 120580. [Google Scholar] [CrossRef]
  321. King, A.; Ndifon, C.; Lui, S.; Widdows, K.; Kotamraju, V.R.; Agemy, L.; Teesalu, T.; Glazier, J.D.; Cellesi, F.; Tirelli, N.; et al. Tumor-homing peptides as tools for targeted delivery of payloads to the placenta. Sci. Adv. 2016, 2, e1600349. [Google Scholar] [CrossRef] [Green Version]
  322. Kaitu’u-Lino, T.J.; Pattison, S.; Ye, L.; Tuohey, L.; Sluka, P.; MacDiarmid, J.; Brahmbhatt, H.; Johns, T.; Horne, A.W.; Brown, J.; et al. Targeted nanoparticle delivery of doxorubicin into placental tissues to treat ectopic pregnancies. Endocrinology 2013, 154, 911–919. [Google Scholar] [CrossRef] [Green Version]
  323. Li, L.; Li, H.; Xue, J.; Chen, P.; Zhou, Q.; Zhang, C. Nanoparticle-Mediated Simultaneous Downregulation of Placental Nrf2 and sFlt1 Improves Maternal and Fetal Outcomes in a Preeclampsia Mouse Model. ACS Biomater. Sci. Eng. 2020, 6, 5866–5873. [Google Scholar] [CrossRef] [PubMed]
  324. EUR-Lex-32007R1394-EN-EUR-Lex. Available online: (accessed on 13 January 2021).
  325. Promising mRNA Tech Comes with Regulatory, CMC Headaches|RAPS. Available online: (accessed on 13 January 2021).
  326. Singh, V.P.; McKinney, S.; Gerton, J.L. Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos. Dev. Cell 2020, 54, 333–347.e7. [Google Scholar] [CrossRef]
  327. Pritchard, N.; Kaitu’u-Lino, T.; Harris, L.; Tong, S.; Hannan, N. Nanoparticles in pregnancy: The next frontier in reproductive therapeutics. Hum. Reprod. Update 2020, 27, 280–304. [Google Scholar] [CrossRef]
  328. Irvin-Choy, N.S.; Nelson, K.M.; Gleghorn, J.P.; Day, E.S. Design of nanomaterials for applications in maternal/fetal medicine. J. Mater. Chem. B 2020, 8, 6548–6561. [Google Scholar] [CrossRef]
  329. Tsukamoto, M.; Ochiya, T.; Yoshida, S.; Sugimura, T.; Terada, M. Gene transfer and expression in progeny after intravenous DNA injection into pregnant mice. Nat. Genet. 1995, 9, 243–248. [Google Scholar] [CrossRef] [PubMed]
  330. Cornford, E.M.; Hyman, S.; Cornford, M.E.; Chytrova, G.; Rhee, J.; Suzuki, T.; Yamagata, T.; Yamakawa, K.; Penichet, M.L.; Pardridge, W.M. Non-invasive gene targeting to the fetal brain after intravenous administration and transplacental transfer of plasmid DNA using PEGylated immunoliposomes. J. Drug Target. 2016, 24, 58–67. [Google Scholar] [CrossRef] [PubMed]
  331. Ellah, N.A.; Taylor, L.; Troja, W.; Owens, K.; Ayres, N.; Pauletti, G.; Jones, H. Development of non-viral, trophoblast-specific gene delivery for placental therapy. PLoS ONE 2015, 10, e0140879. [Google Scholar] [CrossRef]
  332. Giubilato, E.; Cazzagon, V.; Amorim, M.J.B.; Blosi, M.; Bouillard, J.; Bouwmeester, H.; Costa, A.L.; Fadeel, B.; Fernandes, T.F.; Fito, C.; et al. Risk Management Framework for Nano-Biomaterials Used in Medical Devices and Advanced Therapy Medicinal Products. Materials 2020, 13, 4532. [Google Scholar] [CrossRef] [PubMed]
  333. BIOmaterial RIsk MAnagement|BIORIMA Project|H2020|CORDIS|European Commission. Available online: (accessed on 13 January 2021).
  334. Caplan, H.; Olson, S.D.; Kumar, A.; George, M.; Prabhakara, K.S.; Wenzel, P.; Bedi, S.; Toledano-Furman, N.E.; Triolo, F.; Kamhieh-Milz, J.; et al. Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application. Front. Immunol. 2019, 10, 1645. [Google Scholar] [CrossRef]
  335. Bonig, H.; Kuçi, Z.; Kuçi, S.; Bakhtiar, S.; Basu, O.; Bug, G.; Dennis, M.; Greil, J.; Barta, A.; Kállay, K.M.; et al. Children and Adults with Refractory Acute Graft-versus-Host Disease Respond to Treatment with the Mesenchymal Stromal Cell Preparation “MSC-FFM”-Outcome Report of 92 Patients. Cells 2019, 8, 1577. [Google Scholar] [CrossRef] [Green Version]
  336. Namba, F. Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia. Pediatr. Int. 2019, 61, 945–950. [Google Scholar] [CrossRef] [PubMed]
  337. Delhove, J.; Osenk, I.; Prichard, I.; Donnelley, M. Public Acceptability of Gene Therapy and Gene Editing for Human Use: A Systematic Review. Hum. Gene Ther. 2020, 31, 20–46. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  338. Seoane-Vazquez, E.; Shukla, V.; Rodriguez-Monguio, R. Innovation and competition in advanced therapy medicinal products. EMBO Mol. Med. 2019, 11, e9992. [Google Scholar] [CrossRef] [PubMed]
  339. Lipsitz, Y.Y.; Milligan, W.D.; Fitzpatrick, I.; Stalmeijer, E.; Farid, S.S.; Tan, K.Y.; Smith, D.; Perry, R.; Carmen, J.; Chen, A.; et al. A roadmap for cost-of-goods planning to guide economic production of cell therapy products. Cytotherapy 2017, 19, 1383–1391. [Google Scholar] [CrossRef] [PubMed]
  340. Walpole, S.C.; Prieto-Merino, D.; Edwards, P.; Cleland, J.; Stevens, G.; Roberts, I. The weight of nations: An estimation of adult human biomass. BMC Public Health 2012, 12, 439. [Google Scholar] [CrossRef] [Green Version]
  341. Janssen, P.A.; Thiessen, P.; Klein, M.C.; Whitfield, M.F.; Macnab, Y.C.; Cullis-Kuhl, S.C. Standards for the measurement of birth weight, length and head circumference at term in neonates of European, Chinese and South Asian ancestry. Open Med. 2007, 1, e74–e88. [Google Scholar]
  342. Shaw, S.W.S.S.; Blundell, M.P.; Pipino, C.; Shangaris, P.; Maghsoudlou, P.; Ramachandra, D.L.; Georgiades, F.; Boyd, M.; Thrasher, A.J.; Porada, C.D.; et al. Sheep CD34+ amniotic fluid cells have hematopoietic potential and engraft after autologous in utero transplantation. Stem Cells 2015, 33, 122–132. [Google Scholar] [CrossRef]
  343. Aurich, B.; Jacqz-Aigrain, E. Drug safety in translational paediatric research: Practical points to consider for paediatric safety profiling and protocol development: A scoping review. Pharmaceutics 2021, 13, 695. [Google Scholar] [CrossRef]
  344. Ceci, A.; Felisi, M.; Baiardi, P.; Bonifazi, F.; Catapano, M.; Giaquinto, C.; Nicolosi, A.; Sturkenboom, M.; Neubert, A.; Wong, I. Medicines for children licensed by the European Medicines Agency. (EMEA): The balance after 10 years. Eur. J. Clin. Pharmacol. 2006, 62, 947–952. [Google Scholar] [CrossRef]
  345. Giannuzzi, V.; Conte, R.; Landi, A.; Ottomano, S.A.; Bonifazi, D.; Baiardi, P.; Bonifazi, F.; Ceci, A. Orphan medicinal products in Europe and United States to cover needs of patients with rare diseases: An increased common effort is to be foreseen. Orphanet J. Rare Dis. 2017, 12, 64. [Google Scholar] [CrossRef] [Green Version]
  346. TEDDY – European Network of Excellence for Paediatric Clinical Research. European Paediatric Medicines Database (EPMD). Available online: (accessed on 26 October 2021).
  347. Pierce, G.F. Uncertainty in an era of transformative therapy for haemophilia: Addressing the unknowns. Haemophilia 2021, 27, 103–113. [Google Scholar] [CrossRef]
  348. Brooks, S.P.; Bubela, T. Application of protection motivation theory to clinical trial enrolment for pediatric chronic conditions. BMC Pediatr. 2020, 20, 123. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  349. Bushman, F.D. Retroviral Insertional Mutagenesis in Humans: Evidence for Four Genetic Mechanisms Promoting Expansion of Cell Clones. Mol. Ther. 2020, 28, 352–356. [Google Scholar] [CrossRef] [PubMed]
  350. Gene therapy needs a long-term approach. Nat. Med. 2021, 27, 563. [CrossRef] [PubMed]
  351. Blattner, G.; Cavazza, A.; Thrasher, A.J.; Turchiano, G. Gene Editing and Genotoxicity: Targeting the Off-Targets. Front. Genome Ed. 2020, 2, 613252. [Google Scholar] [CrossRef]
  352. Almeida-Porada, G.; Waddington, S.N.; Chan, J.K.Y.; Peranteau, W.H.; MacKenzie, T.; Porada, C.D. In Utero Gene Therapy Consensus Statement from the IFeTIS. Mol. Ther. 2019, 27, 705–707. [Google Scholar] [CrossRef] [Green Version]
Figure 1. Summary of ATMP challenges and potential aggravation or resolution by early intervention. On the right, aggravating influence by early interventions is shown in red; ameliorating influences are in black.
Figure 1. Summary of ATMP challenges and potential aggravation or resolution by early intervention. On the right, aggravating influence by early interventions is shown in red; ameliorating influences are in black.
Pharmaceutics 14 00793 g001
Figure 2. Timeline (prenatal to adult) of ATMP interventions with pros and cons.
Figure 2. Timeline (prenatal to adult) of ATMP interventions with pros and cons.
Pharmaceutics 14 00793 g002
Figure 3. Factors differentially affecting ATMP cost for early vs. adult interventions. Normal black font indicates reduced cost, normal red font indicates increased cost for early interventions.
Figure 3. Factors differentially affecting ATMP cost for early vs. adult interventions. Normal black font indicates reduced cost, normal red font indicates increased cost for early interventions.
Pharmaceutics 14 00793 g003
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Lederer, C.W.; Koniali, L.; Buerki-Thurnherr, T.; Papasavva, P.L.; La Grutta, S.; Licari, A.; Staud, F.; Bonifazi, D.; Kleanthous, M. Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies. Pharmaceutics 2022, 14, 793.

AMA Style

Lederer CW, Koniali L, Buerki-Thurnherr T, Papasavva PL, La Grutta S, Licari A, Staud F, Bonifazi D, Kleanthous M. Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies. Pharmaceutics. 2022; 14(4):793.

Chicago/Turabian Style

Lederer, Carsten W., Lola Koniali, Tina Buerki-Thurnherr, Panayiota L. Papasavva, Stefania La Grutta, Amelia Licari, Frantisek Staud, Donato Bonifazi, and Marina Kleanthous. 2022. "Catching Them Early: Framework Parameters and Progress for Prenatal and Childhood Application of Advanced Therapies" Pharmaceutics 14, no. 4: 793.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop