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Development of AAV-Based Gene Therapies: Unmet Needs and Solutions

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 11008

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Special Issue Editors

Prof. Dr. Stylianos Michalakis

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Guest Editor

Department of Ophthalmology, University Hospital, LMU Munich, Mathildenstraße 8, 80336 Munich, Germany
Interests: gene therapy; retinal degeneration; inherited retinal disorders; adeno-associated virus vectors; preclinical testing; clinical trials; ATMP; viral vector manufacturing; CMC; vector engineering
Special Issues, Collections and Topics in MDPI journals

Dr. Hildegard Buening

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Guest Editor

Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
Interests: gene therapy

Special Issue Information

Dear Colleagues,

Until recently, there were no treatment options for inherited diseases. This has changed with the availability of effective and safe vectors for gene delivery, making gene therapy a valid option for the treatment of inherited diseases. Some of the most promising treatment approaches are gene therapies based on recombinant adeno-associated viruses (AAV). AAV vectors can efficiently deliver genes into somatic cells and lead to the long-term expression of the gene of interest. In addition, they have a favorable safety profile with only low to moderate immunogenicity that is clearly dose-dependent. However, AAV vectors have some limitations that restrict their broad applicability. Due to limited tropism, not all tissues and cells can be reached with existing AAV variants. In addition, there are some diseases caused by mutations in large genes or genes with autosomal dominant inheritance patterns. Further limitations arise from the limited availability of suitable preclinical models that would allow rapid and reliable testing of gene therapy candidates. This Special Issue of IJMS invites submissions of manuscripts addressing such unmet needs for preclinical and/or clinical development of AAV-based gene therapies. These include, but are not limited to, in vitro or in vivo preclinical models, proof-of-concept, validation or safety studies of AAV gene therapy approaches for the treatment of rare or common diseases.

Prof. Dr. Stylianos Michalakis
Dr. Hildegard Buening
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

adeno-associated virus vector
AAV
gene therapy
preclinical testing
analytics
gene delivery
large genes
tropism
autosomal dominant diseases
inherited disorders

Published Papers (6 papers)

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Research

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28 pages, 9638 KiB

Open AccessArticle

RNA-Seq Analysis of Trans-Differentiated ARPE-19 Cells Transduced by AAV9-AIPL1 Vectors

by Alima Galieva, Alexander Egorov, Alexander Malogolovkin, Andrew Brovin and Alexander Karabelsky

Int. J. Mol. Sci. 2024, 25(1), 197; https://doi.org/10.3390/ijms25010197 - 22 Dec 2023

Viewed by 1229

Abstract

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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15 pages, 3350 KiB

Open AccessArticle

Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis

by Meera Vyas, Natalie M. Deschenes, Karlaina J. L. Osmon, Zhilin Chen, Imtiaz Ahmad, Shalini Kot, Patrick Thompson, Chris Richmond, Steven J. Gray and Jagdeep S. Walia

Int. J. Mol. Sci. 2023, 24(19), 14611; https://doi.org/10.3390/ijms241914611 - 27 Sep 2023

Cited by 1 | Viewed by 1053

Abstract

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes—HEXA, HEXB, and GM2A—within the cell’s lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 10¹⁴ vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a^−/−). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a^−/− mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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17 pages, 5432 KiB

Open AccessArticle

Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

by Anett Hudák, Matthew Roach, Dávid Pusztai, Aladár Pettkó-Szandtner, Annamária Letoha, László Szilák, Mimoun Azzouz and Tamás Letoha

Int. J. Mol. Sci. 2023, 24(4), 3141; https://doi.org/10.3390/ijms24043141 - 05 Feb 2023

Cited by 2 | Viewed by 2243

Abstract

Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9’s cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9’s cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9’s cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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Review

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19 pages, 1042 KiB

Open AccessReview

Delivery of Adeno-Associated Virus Vectors to the Central Nervous System for Correction of Single Gene Disorders

by Rrita Daci and Terence R. Flotte

Int. J. Mol. Sci. 2024, 25(2), 1050; https://doi.org/10.3390/ijms25021050 - 15 Jan 2024

Viewed by 1631

Abstract

Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have shown effective adeno-associated virus (AAV) mediated gene transfer for either treatment or prevention of autosomal recessive genetic disorders. Owing to the intricacy of the human CNS and the blood–brain barrier, it is difficult to deliver genes, particularly since the expression of any given gene may be required in a particular CNS structure or cell type at a specific time during development. In this review, we analyzed delivery methods for AAV-mediated gene therapy in past and current clinical trials. The delivery routes analyzed were direct intraparenchymal (IP), intracerebroventricular (ICV), intra-cisterna magna (CM), lumbar intrathecal (IT), and intravenous (IV). The results demonstrated that the dose used in these routes varies dramatically. The average total doses used were calculated and were 1.03 × 10¹³ for IP, 5.00 × 10¹³ for ICV, 1.26 × 10¹⁴ for CM, and 3.14 × 10¹⁴ for IT delivery. The dose for IV delivery varies by patient weight and is 1.13 × 10¹⁵ IV for a 10 kg infant. Ultimately, the choice of intervention must weigh the risk of an invasive surgical procedure to the toxicity and immune response associated with a high dose vector. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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18 pages, 2929 KiB

Open AccessReview

Choroideremia: The Endpoint Endgame

by Maram E. A. Abdalla Elsayed, Laura J. Taylor, Amandeep S. Josan, M. Dominik Fischer and Robert E. MacLaren

Int. J. Mol. Sci. 2023, 24(18), 14354; https://doi.org/10.3390/ijms241814354 - 20 Sep 2023

Cited by 1 | Viewed by 1924

Abstract

Choroideremia is an X-linked retinal degeneration resulting from the progressive, centripetal loss of photoreceptors and choriocapillaris, secondary to the degeneration of the retinal pigment epithelium. Affected individuals present in late childhood or early teenage years with nyctalopia and progressive peripheral visual loss. Typically, by the fourth decade, the macula and fovea also degenerate, resulting in advanced sight loss. Currently, there are no approved treatments for this condition. Gene therapy offers the most promising therapeutic modality for halting or regressing functional loss. The aims of the current review are to highlight the lessons learnt from clinical trials in choroideremia, review endpoints, and propose a future strategy for clinical trials. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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18 pages, 5367 KiB

Open AccessReview

A Review of CRISPR Tools for Treating Usher Syndrome: Applicability, Safety, Efficiency, and In Vivo Delivery

by Lauren Major, Michelle E. McClements and Robert E. MacLaren

Int. J. Mol. Sci. 2023, 24(8), 7603; https://doi.org/10.3390/ijms24087603 - 20 Apr 2023

Cited by 1 | Viewed by 1859

Abstract

This review considers research into the treatment of Usher syndrome, a deaf-blindness syndrome inherited in an autosomal recessive manner. Usher syndrome mutations are markedly heterogeneous, involving many different genes, and research grants are limited due to minimal patient populations. Furthermore, gene augmentation therapies are impossible in all but three Usher syndromes as the cDNA sequence exceeds the 4.7 kb AAV packaging limit. It is, therefore, vital to focus research efforts on alternative tools with the broadest applicability. The CRISPR field took off in recent years following the discovery of the DNA editing activity of Cas9 in 2012. New generations of CRISPR tools have succeeded the original CRISPR/Cas9 model to enable more sophisticated genomic amendments such as epigenetic modification and precise sequence alterations. This review will evaluate the most popular CRISPR tools to date: CRISPR/Cas9, base editing, and prime editing. It will consider these tools in terms of applicability (in relation to the ten most prevalent USH2A mutations), safety, efficiency, and in vivo delivery potential with the intention of guiding future research investment. Full article

(This article belongs to the Special Issue Development of AAV-Based Gene Therapies: Unmet Needs and Solutions)

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