Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups
Abstract
:1. Introduction
2. Concept for In Vivo Organ/Tissue Genome Editing
3. Various Methods and Routes for In Vivo Gene Delivery in Newborn Pups
3.1. Correction of Genetic Disorders through i.v. Introduction of Therapeutic Viral Vectors
3.2. Tissue Tropism among rAAV Serotypes as Revealed by i.v. Introduction of rAAVs to Neonates
3.3. Gene Delivery to Peripheral Tissues through i.v. Introduction of rAAVs in Neonates
3.4. Neonatal i.m. Injection
3.5. Neonatal i.p. Injection
3.6. Gene Delivery via the Retro-Orbital Sinus or toward Retinal Cells
3.6.1. Retinal Gene Delivery into the Subretinal Space
3.6.2. Gene Delivery via the Retro-Orbital Sinus
3.7. Intracerebral Injection
3.8. Gene Delivery to the Skin Cells of Newborn Mice
4. Genome Editing at Neonatal Stages
4.1. Neonatal Gene Correction in Genetic Liver Disorders
4.2. Neonatal Gene Correction in Genetic Blood Clotting Disorders
4.3. Neonatal Gene Correction in Muscular Dysfunction
4.4. Neonatal Gene KO in the CNS
4.5. Clinical Trials Using CRISPR/Cas9-Based Genome Editing
5. Advantages and Limitations of Using Newborns
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AAV | Adeno-associated virus |
ABEs | Adenine base editors |
AD | Adenoviral vector |
APOE3 | Apolipoprotein E3 |
BBB | Blood–brain barrier |
BEs | Base editors |
Cas9 | CRISPR-associated protein 9 |
CBEs | Cytosine base editors |
CNS | Central nervous system |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
dCas9 | Dead Cas9 |
ddPCR | Droplet digital PCR |
DG | Dentate gyrus |
DMD | Duchenne muscular dystrophy |
DRG | Dorsal root ganglia |
DSB | Double-stranded DNA break |
EGFP | Enhanced green fluorescent protein |
ENS | Enteric nervous system |
EP | Electroporation |
ER | Estrogen receptor |
ERT2 | Mutated ligand-binding domain of ER |
ET | Egg transfer |
F8 (9) | Factor 8 (9) |
GAG | Glycosaminoglycan |
GALC | β-Galactocerebrosidase |
GCs | Genome copies |
GFP | Green fluorescent protein |
GOI | Gene of interest |
GONAD/i-GONAD | Genome Editing via Oviductal Nucleic Acids Delivery/improved GONAD |
gRNA | Guide RNA |
GUSB | β-Glucuronidase |
HDR | Homology-directed repair |
HA | Hemophilia A |
HB | Hemophilia B |
HGD | Hydrodynamics-based gene delivery |
ICV | Intracerebroventricular |
IDUA | α-L-iduronidase |
i.m. | Intramuscular |
i.p. | Intraperitoneal |
IT | Intrathecal |
ITRs | Inverted terminal repeats |
i.v. | Intravenous |
KI | Knock-in |
KO | Knockout |
LMNs | Lower motor neurons |
LPS | Lipopolysaccharide |
LV | Lentivirus |
MMEJ | Microhomology-mediated end-joining |
MPS I | Mucopolysaccharidosis type I |
MPS VII | Mucopolysaccharidosis type VII |
NCAM (hNCAM-140) | Neural cell adhesion molecule |
nCas9 | Cas9 nickase |
NF-κB | Nuclear factor-κB |
NHEJ | Non-homologous end-joining |
NSCs | Neural stem cells |
OB | Olfactory bulb |
4OHT | 4-Hydroxytamoxifen |
OTC | Ornithine transcarbamylase |
PAM | Protospacer adjacent motif |
PBS | Phosphate-buffered saline |
PEs | Prime editors |
pegRNA | Prime editing guide RNA |
PFV | Foamy virus |
PG | Periglomerular |
RGCs | Radial glial cells |
RMS | Rostral migratory stream |
RNAi | RNA interference |
RNP | Ribonucleoprotein |
RT | Reverse transcriptase |
RV | Retrovirus |
rAAV | Recombinant adeno-associated virus |
SaCas9 | Staphylococcus aureus-derived Cas9 |
SB | Sleeping Beauty |
SFFV | Spleen focus-forming virus |
sgRNA | Single-guide RNA |
shRNA | Short hairpin RNA |
siRNAs | Short interfering RNAs |
SN | Substantia nigra |
Sod1 | Superoxide dismutase 1 |
SVZ | Subventricular zone |
TadA | tRNA adenine deaminase |
TBG | Thyroxine-binding globulin |
Tg | Transgenic |
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Route and Method of Gene Delivery | Type of Nucleic Acid(s) Introduced | Outcome | Target Gene or Gene of Interest (GOI) Introduced | References |
---|---|---|---|---|
Facial-vein-mediated injection (also called i.v. injection) | Recombinant adeno-associated virus (rAAV) | Single injection of rAAV carrying human beta-glucuronidase (GUSB) cDNA into mucopolysaccharidosis type VII (MPS VII) model mice resulted in the expression of GUSB in most organs for 16 weeks and decreased or complete prevention of lysosomal storage; particularly, cells in the CNS were cleared of disease, suggesting viral infection beyond the blood–brain barrier (BBB) | GUSB | Daly et al., 1999 [66] |
Facial-vein-mediated injection (also called i.v. injection) | Retroviral vector (RV) | Neonatal injection of RV carrying canine GUSB cDNA into dogs with MPS VII resulted in clonal expansion of hepatocytes and secretion of active GUSB into serum, along with clinical improvements; this is the first successful application of gene therapy in preventing a lysosomal storage disease in a large animal | GUSB | Ponder et al. 2002 [67] |
Facial-vein-mediated injection (also called i.v. injection) | RV | Neonatal injection of RV carrying canine GUSB cDNA into MPS VII mice resulted in transduction of 6 to 35% of hepatocytes, which secreted GUSB into the blood; the secreted enzyme was taken up by other tissues and reduced the histopathological evidence of lysosomal storage in the liver, spleen, kidneys, small intestine, neurons, and glial cells | GUSB | Xu et al., 2002 [68] |
Facial-vein-mediated injection (also called i.v. injection) | Adenoviral vector (AD) | Neonatal single injection of AD carrying human GUSB cDNA into MPS VII mice resulted in the recovery of more than 20% of GUSB activity in the brain, leading to the prevention of lysosomal storage, and lacking characteristic facial skeletal deformities associated with bone deformity, mental retardation, corneal clouding, and retinal degeneration | GUSB | Kamata et al., 2003 [69] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2 | Neonatal i.v. injection of rAAV2 carrying human iduronidase (IDUA) cDNA was performed using newborns with mucopolysaccharidosis type I (MPS I) to determine the potential for the gene delivery approach; high levels of IDUA activity were present in the treated animals and persisted for the 5-month duration of the study; successful correction of metabolic, craniofacial, and neurological abnormalities in MPS I mice was achieved | IDUA | Hartung et al., 2004 [70] |
Facial-vein-mediated injection (also called i.v. injection) | Lentiviral vector (LV) | Single injection of LV into MPS I model mice resulted in the expression of IDUA activity, decreased glycosaminoglycan (GAG) storage, prevention of skeletal abnormalities, a more normal gross appearance, and improved survival; most strikingly, transduction of neurons at high levels was prominent | IDUA | Kobayashi et al., 2005 [71] |
Peripheral (tail vein) i.v. injection | rAAV2/8 rAAV2/9 | Both rAAV2/8 and rAAV2/9 vectors caused substantial transduction in the heart, skeletal muscle, and pancreas; importantly, rAAV2/9 transduced myocardium 5–10-fold higher than rAAV2/8, resulting in over 80% cardiomyocyte transduction, and suggesting that rAAV2/9 is superior to rAAV2/8 specifically for cardiac gene delivery | F9 nlslacZ lacZ | Inagaki et al., 2006 [72] |
Facial-vein-mediated injection (also called i.v. injection) | Sleeping Beauty (SB) transposon | Newborn mice were first i.p. injected with mannitol, and 1 h later luciferase-transposon DNA conjugated with polyethylenimine (PEI) was injected into the lateral ventricle; the treated animals showed significantly higher luciferase expression one month after gene delivery, suggesting chromosomal integration of the luciferase transgene and the usefulness of mannitol pretreatment combined with transposon-mediated gene transfer for long-term gene expression in the mammalian brain | Luciferase | Demorest et al., 2006 [58] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2/1 rAAV2/8 rAAV2/9 | When comparing rAAV2/1 with rAAV2/8 and the newer rAAV2/9 vectors for the transduction of skeletal muscle, both rAAV2/8 and rAAV2/9 were able to transduce myocardium at approximately 20- and 200-fold (respectively) higher levels than rAAV2/1; thus, rAAV2/9 is more readily able to cross the vasculature and leads to preferential cardiac transduction in vivo, efficiently transducing cardiac tissue | lacZ | Pacak et al., 2006 [73] |
Facial-vein- or external jugular-vein-mediated injection (also called i.v. injection) | - | Two techniques (injection via the external jugular vein, and via the superficial temporal vein) for i.v. injection in neonatal mice from birth to 6 days of age were described; both allow the injection of a variety of substances, including cells, medications, toxins, and cytokines, and both permit serial injections | - | Kienstra et al., 2007 [60] |
Facial-vein-mediated or intraperitoneal (i.p.) injection | rAAV8 | Neonatal administration of rAAV8 by i.v. or i.p. injection was performed to test whether lower motor neurons (LMNs) are transduced, indicating that dorsal root ganglion transduction occurs across all timepoints and injection routes | GFP | Foust et al. 2008 [74] |
Facial-vein-mediated injection (also called i.v. injection) | Helper-dependent adenoviral vector (AD) | Intravenous (i.v.) injection of AV expressing human factor 8 (F8) to neonatal hemophilia A (HA)-knockout (KO) mice resulted in the production of high levels of F8, and its expression lasted until >1 y of age, which is associated with correction of HA and tolerance to human F8 | F8 | Hu et al., 2011 [75] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2/9 | Neonatal administration of rAAV2/9 produced global delivery to the central (i.e., brain, spinal cord, and all layers of the retina) and peripheral nervous system (i.e., myenteric plexus and innervating nerves), which can provide a therapeutic strategy for the treatment of early lethal genetic diseases, such as Gaucher disease | GFP | Rahim et al. 2011 [76] |
Facial-vein-mediated injection (also called i.v. injection) | rAAVrh10 | Neonatal co-injection of two AAVrh10s (in which one had a heavy chain and the other a light chain for F8) into mice with HA resulted in long-term correction and avoidance of immune responses in the AAV-F8-treated mice | F8 | Hu and Lipshutz 2012 [77] |
i.v.-mediated injection | rAAV9 carrying mutating capsid surface tyrosines | A double-tyrosine mutant of rAAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression could be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter, which may provide a new methodology for the development of retinal gene therapies or the creation of animal models of neurodegenerative diseases | GFP | Dalkara et al., 2012 [78] |
Facial-vein-mediated injection (also called i.v. injection) | Self-complementary (sc) rAAV9 | Neonatal i.v. injection of rAAV9 resulted in gene transfer to all layers of the retina (including retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells, and retinal ganglion cells) in adult mice; in particular, the cells on the inner side of the retina were transduced with the highest efficiency, suggesting that this vector serotype is able to cross mature blood–eye barriers | GFP | Bemelmans et al., 2013 [79] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2/1 rAAV2/5 rAAV2/6 rAAV2/8 rAAV2/9 | Intravenous (i.v.) injection of AAV9 resulted in the transduction of 25–57% of enteric nervous system (ENS) myenteric neurons; AAV9 transduction in enteric glia was very low compared to CNS astrocytes; AAV8 resulted in comparable transduction in neonatal mice to AAV9, while AAV1, -5, and -6 were less efficient, suggesting that systemic AAV9 has a high affinity for peripheral neural tissue and will be useful for future therapeutic development and basic studies of the ENS | GFP | Gombash et al., 2014 [80] |
Facial-vein-mediated injection (also called i.v. injection) | Single-stranded (ss) and scAAV9 | Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced, suggesting that AAV9 can potentially be used for clinical systemic gene therapy protocols | GFP | Mattar et al., 2015 [81] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2/8 rAAV2/9 | Both rAAV2/8 and rAAV2/9 showed equal potential in transducing the ENS, with 25–30% of the neurons expressing EGFP; all enteric neuron subtypes, but not glia, expressed the reporter protein; these findings will be used for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and for gene therapy in the longer term | EGFP | Buckinx et al., 2016 [82] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV2/9 | Resulted in binaural transduction of inner hair cells, spiral ganglion neurons, and vestibular hair cells; transduction efficiency increased in a dose-dependent manner; inner hair cells were transduced in an apex-to-base gradient, with transduction reaching 96% in the apical turn; intravenous delivery of rAAV2/9 represents a novel and atraumatic technique for inner-ear transgene delivery in early postnatal mice | EGFP | Shibata et al., 2017 [83] |
Facial-vein-mediated injection (also called i.v. injection) or intracerebral injection | Foamy virus (PFV) | Systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression; transgene expression was highly localized to the hippocampal architecture, despite vector delivery being administered to the lateral ventricle; PFV can be used for neonatal gene delivery targeted to hippocampal neurons, for gene therapy of neurological disorders | EGFP | Counsell et al., 2018 [84] |
Facial-vein-mediated injection (also called i.v. injection) | AAVs | Intravenous (i.v.) administration can achieve widespread delivery of rAAVs to the CNS, which are considered to be promising therapeutic tools for treating genetic defects of the CNS, due to their excellent safety profile and ability to cross the BBB | - | Gessler et al., 2019 [85] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV8 | Intravenous (i.v.) administration can achieve widespread gene expression in the central and peripheral nervous system, liver, kidneys, and skeletal muscle; i.v. injection of rAAV8 carrying a spleen focus-forming virus (SFFV) promoter and nuclear factor-κB (NF-κB) binding sequence for bioluminescence and biosensor evaluation resulted in a 10-fold increase in luc expression after single administration of lipopolysaccharide (LPS), and whole-body bioluminescence persisted for up to 240 days | GFP luc | Karda et al., 2020 [6] |
Intracerebral injection | rAAV2 | Gene transfer throughout the CNS was achieved without germline transmission, and gene expression lasted for at least 1 year | GUSB | Passini and Wolfe 2001 [86] |
Intracerebral injection | rAAV1 rAAV2 rAAV5 | The 0gene delivery pattern after neonatal intracerebral injection was assessed using different rAAV serotypes; consequently, rAAV5 showed very limited brain transduction, even though it has different transduction patterns than rAAV2; in contrast, rAAV1 vectors showed robust widespread transduction; in the majority of structures, rAAV1 transduced many more cells than rAAV2 | GUSB | Passini et al., 2003 [87] |
Intracerebral injection and subsequent in vivo electroporation (EP) | Plasmid DNA | Intraventricular injection of DNA followed by EP induced strong expression of transgenes in the radial glia, neuronal precursors and neurons of the olfactory system; overexpression of the cell-cycle inhibitor p21 resulted in interference with the proliferation of neural stem cells | hNCAM-140 p21 | Boutin et al., 2008 [88] |
Intracerebral injection and subsequent in vivo EP | Plasmid DNA | Neonatal intracerebral injection was performed to determine how neuroprogenitors in the subventricular zone (SVZ) give rise to neuroblasts that migrate along the rostral migratory stream (RMS); labeling was found in all classes of interneurons in the olfactory bulb (OB), persisted to adulthood, and had no adverse effects | EGFP | Chesler et al., 2008 [89] |
Intracerebral injection | rAAV8 LV | Region-specific recombination of a “stop-floxed” Rosa26 reporter allele was achieved upon targeted injection of rAAV vectors expressing Cre-recombinase (Cre); utilizing LV, efficient transduction of neuroprogenitors in the SVZ occurred and, as a result, approximately 20% of labeled migrating neuroblasts were generated along the RMS into the OB | Cre | Pilpel et al., 2009 [90] |
Intracerebral injection and subsequent in vivo EP | Plasmid DNA | Neonatal non-ventricular injection into deep cortical layers or the striatum region, followed by EP, was performed to create a local expression pattern in the area of interest and in situ transfection of non-migratory cell types, e.g., cortical astrocytes, which may be used for two-photon in vivo imaging | EGFP | Molotkov et al., 2010 [91] |
Intracerebral injection and subsequent in vivo EP | Plasmid DNA | Improvement of the EP technique allowing for targeted transgene delivery to specific walls of the lateral ventricle, accurately and reproducibly, was performed to trace perinatal neural stem cells, which successfully enabled fate mapping of the progeny of RGCs | EGFP | Fernandez et al., 2011 [92] |
Intracerebral injection | rAAV8 | Neonatal intra-cerebral injection of viral vectors into the hindbrain enables postnatal dendritic maturation in cerebellar Purkinje neurons for in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction | YFP tdTomato iCre tTA | Kim et al., 2013 [93] |
Intracerebral injection | rAAV2/1 rAAV2/2 rAAV2/5 rAAV2/7 rAAV2/8 rAAV2/9 | The effects of the timing of the injection on the rAAV tropism and biodistribution of six commonly used rAAVs after neonatal intracerebral gene delivery was assessed; consequently, rAAV2/8 and 2/9 resulted in the most widespread distribution in the brain; injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration at later periods of development resulted in more non-neuronal transduction; rAAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection; none of the serotypes tested showed any microglial transduction | EGFP | Chakrabarty et al., 2013 [94] |
Intracerebral injection | LV | Neonatal intracerebral injection of LV carrying the β-galactocerebrosidase (GALC) gene in the external capsule of twitcher mice, a severe model of globoid cell leukodystrophy, resulted in the restoration of GALC activity in the whole CNS of treated mice as early as 8 days post-injection; this approach will be useful for neonatal LV-mediated intracerebral gene therapy | GALC GFP | Lattanzi et al., 2014 [95] |
Intracerebral injection and subsequent in vivo EP | Plasmid DNA | Neonatal intracerebral injection of plasmid and subsequent in vivo EP were performed to analyze the RMS and postnatal OB neurogenesis; consequently, GFP-positive cells in the dentate gyrus (DG) were observed to extend branched dendrites and long axons into the molecular layer and the hilus; the expression of GFP in these neurons was sustained for at least 9 months | GFP | Ito et al., 2014 [96] |
Intracerebral injection | rAAV8 | Intraventricular injection of rAAV8 within the first 24 h after birth resulted in widespread transduction of neurons throughout the brain; expression began within days of the injection and persisted for the lifetime of the animal; this versatile manipulation enables studies ranging from early postnatal brain development to aging and degeneration in the adult | YFP tdTomato | Kim et al., 2014 [97] |
Intracerebral injection | rAAV2/1 rAAVDJ8 rAAV9 | Tropism of rAAV2/1, rAAVDJ8, and rAAV9 throughout different brain regions and cell types was assessed through neonatal intracerebral injection; rAAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than rAAVDJ8 and rAAV9; rAAVDJ8 displayed more tropism in astrocytes compared to rAAV9 in the substantia nigra (SN) region | GFP | Hammond et al., 2017 [98] |
Intracerebral injection | rAAV9 AAV-PHP.B AAV-PHP.eB | Neonatal intra-brain injection of rAAV9, AAV-PHP.B, and AAV-PHP.eB carrying CRISPR reagents was used to examine cell-type-specific gene ablation; consequently, AAV-PHP.B variants exhibited marked disruption of neuron-related genes, but only modest disruption of the astrocyte- or oligodendrocyte-specific genes was observed by all three AAV variants, which could facilitate profiling of AAV cellular tropism in the murine CNS | NeuN GFAP MOG | Torregrosa et al., 2021 [99] |
Intraperitoneal (i.p.) injection | rAAV1 rAAV2 rAAV5 rAAV6 rAAV7 rAAV8 | Neonatal single injection of various serotypes of rAAVs via i.p. or i.v. routes was performed; rAAV8 was the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles; rAAV1 and rAAV6, which demonstrated robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier; gene expression persisted in the muscle and heart but diminished in the liver, which showed rapid cell division; this approach will be useful for muscle-directed systemic gene therapy | GFP | Wang et al., 2005 [100] |
i.p. injection | Single-stranded AAV9 (ssAAV9) carrying short hairpin RNA (shRNA) | Neonatal i.p. injection of shRNA-ssAAV9 resulted in ~80% reduction in target mRNA in the DRG, along with 75% suppression of the protein; the suppression effect lasted for more than 3 months; this approach may be helpful for elucidating the mechanisms of pain and sensory ganglionopathies | Sod1 | Machida et al., 2013 [64] |
i.p. and i.v. injection | rAAV8 | Neonatal i.p. or i.v. injection of rAAV8 through entry into the nervous system was performed to target lower motor neurons (LMNs) for future gene therapy against spinal muscular atrophy and amyotrophic lateral sclerosis; consequently, spinal cords were positively transduced; furthermore, fibers in the dorsal horns and columns were labeled, indicating dorsal root ganglion transduction with these techniques | GFP | Foust et al., 2008 [74] |
i.v. injection via the retro-orbital sinus | RV | Neonatal retro-orbital-sinus-mediated injection of an RV vector carrying F8 cDNA into F8-deficient mice resulted in successful correction of HA | F8 | Vanden Driessche et al., 1999 [101] |
Injection into the subretinal space and subsequent in vivo EP | Plasmid DNA | Neonatal injection of plasmid DNA into the subretinal space of mice, and subsequent in vivo EP, resulted in successful transfection of retinal cells; transfection of plasmid DNA carrying RNAi resulted in a knockdown phenotype of a target gene’s expression, which was similar to the phenotype shown in KO mice | GFP | Matsuda and Cepko 2004 [102] |
Injection into the subretinal space and subsequent in vivo EP | Plasmid DNA | Conditional temporal and spatial regulation of gene expression in the retinas of postnatal rats was assessed using Cre/loxP-mediated inducible expression vectors and 4-hydroxytamoxifen treatment, which enables conditional activation of Cre recombinase; transgene expression was successfully induced in a cell-type- and time-specific manner | CreERT2 DsRed GFP | Matsuda and Cepko 2007 [103] |
Hydrodynamics-based gene delivery (HGD) via the retro-orbital sinus | Plasmid DNA | High levels of gene expression in the hepatocytes of neonatal mice were achieved, which will provide a way to perform gene delivery to animals that are difficult to inject via the tail vein; it will also be beneficial for exploring gene function and treating genetic disease | Luciferase | Yan et al., 2012 [104] |
i.v. injection via the retro-orbital sinus | First-generation AD vector | The highest AD vector genome copy numbers and transgene expression were found in the neonatal liver; the neonatal heart exhibited the second-highest levels of transgene expression among the organs examined; no apparent hepatotoxicity was observed in neonatal mice; these findings may be helpful for performing gene therapy using AD vectors in neonates | Luciferase | Iizuka et al., 2015 [105] |
i.v. injection via the retro-orbital sinus | rAAV9 | Showing the benefits of i.v. injection via the retro-orbital sinus as an effective route of rAAV9-mediated gene delivery in neonates | Cre | Prabhakar et al., 2021 [59] |
Intramuscular (i.m.) injection | rAAV | Neonatal i.m. injection of an rAAV vector carrying human GUSB cDNA into MPS VII mice resulted in high-level intramuscular GUSB expression as early as 2 weeks of age, and for at least 16 weeks; GUSB activity was detected in both the liver and spleen at later timepoints, indicating that rAAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth | GUSB | Daly et al., 1999 [106] |
i.m. injection | Plasmid DNA | Neonatal intramuscular injection of plasmid DNA into hypercholesterolemic mice (ApoE KO mice) resulted in a reduction in the incidence of severe hypercholesterolemia; notably, when naked DNA was administrated early, no immune response was generated against the human APOE3, allowing for repeated administrations; this approach will be useful for treating many genetic childhood diseases where early administration is required to prevent developmental damage | APOE3 | Signori et al., 2007 [107] |
Injection into the skin and subsequent in vivo EP | Plasmid DNA | Subcutaneous injection of two plasmid DNAs (one with a neomycin-resistance gene (neo) and the other with an immortalizing gene) into the skin cells of newborn mice (at P1-3), followed by subsequent in vivo EP, resulted in the generation of stably transfected fibroblasts | neo | Titomirov et al., 1991 [65] |
Route and Method of Gene Delivery | Type of Nucleic Acid(s) Introduced | Outcome | Target Gene or Gene of Interest (GOI) Introduced | References |
---|---|---|---|---|
Facial-vein-mediated injection (also called i.v. injection) | Dual rAAV (one with the Cas9 gene and the other with gRNA/donor DNA) | Neonatal i.v. injection of dual AAVs into OTC-deficient mice was performed to correct metabolic liver disease caused by lethal hyperammonemia; consequently, 10% of hepatocytes were restored, and reduced survival after feeding with a chow diet was avoided | OTC | Yang et al., 2016 [118] |
Facial-vein-mediated injection (also called i.v. injection) | Dual rAAV8 (one with SaCas9/sgRNA and the other with donor DNA) | Neonatal i.v. injection of an SaCas9/sgRNA-expressing rAAV8 vector into wild-type mice resulted in mutations in the F9 gene in hepatocytes, sufficiently developing HB; also, it was possible to generate HDR-based correction of the mutated F9 gene in HB model mice; this approach will provide a flexible approach to induce DSB-mediated mutations in target genes in hepatocytes, and also to cure congenital hemorrhagic disease | F9 | Ohmori et al., 2017 [119] |
Facial-vein-mediated injection (also called i.v. injection) | Dual rAAV8 (one with SaCas9 and the other with gRNA/donor DNA containing F9 cDNA carrying a hyperactive F9 Padua mutation | Neonatal i.v. injection of dual rAAVs into F9 KO mice resulted in expression of F9 at a normal level over 8 months, suggesting the use of the CRISPR approach to achieve lifelong expression of therapeutic proteins | F9 | Wang et al., 2019 [120] |
Facial-vein-mediated injection (also called i.v. injection) | rAAV8 carrying donor DNA and rAAV8 carrying SaCas9 and sgRNA | Neonatal i.v. injection of dual rAAV8 vectors into F9 KO mice resulted in the stable expression of human F9, reaching up to 150% of the human levels, showing a clotting capacity comparable to wild-type animals, and demonstrating the rescue of the disease phenotype | F9 | Lisjak et al., 2022 [121] |
Intracerebral injection | AAV-PHP.B carrying sgRNA | Neonatal intracerebral injection of AAV-PHP.B carrying sgRNA into Cas9 mice resulted in a 99.4% rate of biallelic indels in the transduced cells, leading to a more than 70% reduction in the quantity of total NeuN proteins in the cortex, hippocampus, and spinal cord | NeuN | Hana et al., 2021 [122] |
i.p. injection | Dual rAAV9 carrying CRISPR components | To correct Duchenne muscular dystrophy (DMD) by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, rAAV9 carrying CRISPR/Cas9 components was i.p. injected into neonatal mdx mice (P1), a model of DMD; as a result, dystrophin protein expression was observed in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after te injection | Dystrophin | Long et al., 2016 [123] |
i.m. injection | Dual rAAV8 carrying CRISPR components | Neonatal i.m. injection of rAAV8 carrying CRISPR components into an mdx mouse model of DMD was performed to improve muscle function; removal of mutated exon 23 from the dystrophin gene resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, and significant enhancement of muscle force, suggesting that this approach is useful as a potential therapy to treat DMD | Dystrophin | Nelson et al., 2016 [124] |
i.m. injection | Dual rAAV9 carrying CRISPR components | To test whether the removal of one or more exons from the mutated transcript in DMD-related dystrophin could produce an in-frame mRNA and a truncated but still functional protein, dual rAAVs carrying CRISPR/Cas9 components were subjected to i.m. injection into neonatal mdx model mice; as a result, in the treated mice, restoration of dystrophin expression was observed in myofibers, cardiomyocytes, and muscle stem cells | Dystrophin | Tabebordbar et al., 2016 [125] |
Procedure or Property | Newborns | Adults |
---|---|---|
Anesthesia | Relatively easy, because newborn pups are prone to hypothermia after being placed on a chilled paper towel on top of ice for 30–60 s; therefore, there is no need for an anesthetic agent, but the surgical procedure must be performed within ~30 min | Requires administration of an anesthetic agent or isoflurane; surgical procedure can be performed for more than 1 h |
Immunological tolerance | The immune system of newborn pups at P0 to P7 (whose condition is also called “immune immaturity”) is not well established; therefore, it is possible to transplant xenogenic cells such as human-derived cells | The immune system is already tightly established; therefore, it is impossible to transplant xenogenic cells such as human-derived cells |
Intravenous (i.v.) injection | Relatively easy; a small amount of solution (up to 50 μL) can be injected via the facial vein; therefore, the reagents required can be reduced at a minimal level; however, the appearance of the facial vein is temporal, as it disappears up to 6 days after birth; tail vein injection becomes possible from P15 onward; therefore, it may be theoretically impossible to perform i.v. injection during the periods of P7 to P14 | Relatively easy; relatively large amounts of solution (~1 mL) are generally required; therefore, the reagents used appear to be costly |
Gene delivery beyond the blood–brain barrier (BBB) after i.v. injection | Due to the incomplete development of the BBB at this stage, small molecules such as recombinant adeno-associated viruses (rAAVs) injected exogenously can easily penetrate into cells of the central nervous system (CNS) via the BBB | Large molecules cannot be transferred inside a brain, since the established BBB does not allow penetration beyond the BBB |
Intracerebral injection | Relatively easy; requires peeling of the skin and skull with fine forceps prior to the insertion of a capillary pipette under a dissecting microscope | It requires drilling of a skull prior to the insertion of the capillary pipette, which is laborious and time-consuming |
Retro-orbital-sinus-mediated injection | Relatively easy | Relatively easy; repeated treatment is possible |
Manipulation of internal organ | Relatively difficult, because organs are too small and fragile; therefore, their handling must be carried out under observation using a dissecting microscope, requiring special caution upon surgical treatment and skin closure after surgery, because the skin itself is thin and labile | Relatively easy; manipulation of internal organs can be performed during their exposure onto the skin; also, skin closure is easy after surgery |
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Nakamura, S.; Morohoshi, K.; Inada, E.; Sato, Y.; Watanabe, S.; Saitoh, I.; Sato, M. Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups. Int. J. Mol. Sci. 2023, 24, 15301. https://doi.org/10.3390/ijms242015301
Nakamura S, Morohoshi K, Inada E, Sato Y, Watanabe S, Saitoh I, Sato M. Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups. International Journal of Molecular Sciences. 2023; 24(20):15301. https://doi.org/10.3390/ijms242015301
Chicago/Turabian StyleNakamura, Shingo, Kazunori Morohoshi, Emi Inada, Yoko Sato, Satoshi Watanabe, Issei Saitoh, and Masahiro Sato. 2023. "Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups" International Journal of Molecular Sciences 24, no. 20: 15301. https://doi.org/10.3390/ijms242015301