Human gene therapy最新文献

CLN5 disease, caused by mutations in the CLN5 gene, is a form of neuronal ceroid lipofuscinoses (Batten disease). Patients suffer progressive motor dysfunction, vision loss, seizures, and dementia, leading to premature death. Here, we report a preclinical study of AAV9-mediated gene therapy in a Cln5^-/- mouse model. Single-dose AAV9 carrying human CLN5 driven by the CAG or human synapsin 1 promoter (hSYN) was administered via intracerebroventricular injection into neonatal and juvenile Cln5^-/- mice. Treatment efficacy was evaluated by assessment of neurodegeneration, neuroinflammation, locomotor function, and survival. AAV9 expressing CLN5 driven by the hSYN promoter significantly alleviated neurodegeneration, improved biochemical and glycosphingolipid profiles, neuropathological and locomotor function, and extended lifespan of the Cln5^-/- mice. However, gene transfer employing the CAG promoter demonstrated limited therapeutic efficacy. Furthermore, delayed intervention in juveniles provided superior therapeutic response compared with early neonatal intervention and normalized lifespan. Finally, blood plasma neurofilament light that is significantly elevated in the Cln5^-/- mice is restored to normal wildtype levels following treatment. These results indicate that brain-directed adeno-associated virus (AAV) gene therapy could be a promising treatment strategy for CLN5 disease and efficacy might be monitored using a noninvasive blood plasma biomarker.

Recombinant adeno-associated viruses (rAAVs) are useful vectors for clinical gene therapy. It is crucial to examine DNA impurities, such as plasmid DNA, for quality control of rAAV products. In this study, we examined highly purified rAAV1, rAAV2, rAAV5, and rAAV6 samples produced on a three-plasmid platform, using a high-throughput sequencer. These samples contained 0.49-3.80% detectable DNA impurities derived from the three plasmids, as estimated by the ampicillin resistance gene (amp^R). The plasmid impurities consisted of 90.62-95.84% pAAV, 3.21-6.83% pRC, and 0.95-2.55% pHelper DNA. These trends were consistent with those of DNA impurities determined by droplet digital PCR (ddPCR), indicating that cleaved pAAV backbone DNA was the primary source of DNA impurities encapsulated into the capsids. To examine the preferential encapsulation of pAAV backbone DNA into capsids, short sequencing reads were mapped to the entire pAAV backbone sequence, and we found that the reads were relatively evenly distributed across the backbone sequence, with occasional sharp drops. Furthermore, the read length distribution containing the pAAV backbone sequence showed a main peak at 3.2 kb in Oxford Nanopore Technologies sequencing. This length was consistent with that of backbone DNA nicked at two terminal resolution sites by Rep78/68. Analysis of the terminal sequences of the long reads containing backbone sequences revealed that 92.7-98.4% of them contained Rep-binding elements. These results indicate that the pAAV backbone in linear DNA form was cleaved from pAAV-ZsGreen1 by Rep78/68 nicking at trs in the nucleus, and that the single-stranded DNA was efficiently translocated into the capsids through the encapsulating machinery, similar to the rAAV genome.

Systemic delivery of adeno-associated viral (AAV) vectors is a promising approach for brain gene therapy, particularly in combination with emerging techniques such as focused ultrasound (FUS), which can transiently and noninvasively open the blood-brain barrier to facilitate delivery of AAVs to the brain. However, off-target vector accumulation, particularly in the liver, remains a significant safety concern. To address this, we introduced a mutation into variable region 1 of the AAV9 capsid (G266A) and assessed its effect on liver and brain transduction, testing both direct intraparenchymal injection and intravenous injection with FUS. Interestingly, we found that G266A mutation strongly decreased liver transduction while having only a modest effect on transduction of the brain and other tissues. We also found that the G266A mutation had minimal impact on cell attachment and uptake but strongly decreased binding of the capsid to both human and mouse AAV receptor (AAVR), which likely explains the decrease in liver transduction. Overall, our findings suggest that the G266A mutation and modification of AAVR binding could be a useful strategy to mitigate liver toxicity associated with systemic brain gene therapy.

Epigenome editing technology holds great promise for treating diverse genetic disorders. In this study, we demonstrate epigenetic activation of the LAMA1 gene for the treatment of LAMA2-CMD, a severe congenital muscle dystrophy (CMD) caused by biallelic mutations in the LAMA2 gene. LAMA1 is a sister homolog that is known to compensate for the function of LAMA2. However, supplementing LAMA1 or LAMA2 gene via viral platform is not feasible due to the large size of their coding sequences. Through a single administration of our adeno-associated virus (AAV) vector encoding all the necessary elements for epigenetic activation, we observed significant LAMA1 gene upregulation and phenotype improvements in mouse disease models. The muscle-tropic AAV capsid exhibited desired vector biodistribution and promising pharmacodynamics with good safety profiles in 2-year-old juvenile nonhuman primates (NHPs). Moreover, administration to 8-month-old infant NHPs demonstrated superior pharmacodynamics compared with 2-year-old juveniles, even at half the dose. Our approach holds broad applicability for a range of loss-of-function genetic disorders and could offer a therapeutic breakthrough where active epigenome offers clinical benefit.

Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer's disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3.5-fold in E3/E4 heterozygotes and 15-fold in E4 homozygotes. Since the E3 and E4 alleles differ only by a single nucleotide, gene editing of E4 to E3 is a potential strategy to reduce AD risk in E4 homozygotes. Because the APOE pool in the brain is separate from systemic APOE, editing to treat AD would ideally be directed to the brain. Following in vitro optimization of prime editing guide RNAs, efficient prime editing expression cassettes were inserted into the adeno-associated virus (AAV) split-intein system and packaged into pairs of AAV vectors for in vivo editing. The AAV vectors were administered to human homozygous APOE4-targeted replacement mice (TRE4), and APOE4 to APOE3 editing efficiency was assessed after 4 weeks. The prime editing construct designated APOE3/4-3_10 was the most efficient at APOE4 to APOE3 conversion, both in liver following intravenous delivery and in brain following intrahippocampal delivery. To assess brain-wide editing, two AAV capsids were compared, including AAVrh.10 with administration either directly to the hippocampus or to the cerebrospinal fluid via the cisterna magna and AAV-CAP.B10 administered intravenously. Other than minor differences in APOE4/3-3_10 mediated E4 to E3 editing in the cerebellum, the different capsids and routes yielded similar editing efficacy throughout the brain. This may represent a candidate treatment to reduce the risk of AD.

Polyethylenimine (PEI) is widely employed as a transfection reagent in recombinant adeno-associated virus (rAAV) manufacturing, but it must be removed from the final product due to its potential toxicity. Accurate quantification of PEI in complex biological matrices such as rAAVs is challenging, largely because the strong electrostatic attraction between PEI and nucleic acids can hinder the accuracy of its quantification. Here, we report a robust high-performance liquid chromatography method with charged aerosol detection for the quantification of residual linear PEI in purified AAV samples. Sample preparation includes treatment with trifluoroacetic acid and hydrochloric acid at 60°C to denature capsid protein, disrupt PEI-DNA polyplexes, and hydrolyze nucleic acids. The method achieves a limit of detection of 5 µg/mL and a limit of quantitation of 10 µg/mL in spike-and-recovery studies, with quantification confirmed via visual peak identification. This approach enables sensitive, specific, and reproducible PEI measurement and provides a valuable tool for process monitoring and quality control in gene therapy manufacturing.

Preliminary investigations focused on biodistribution of polymeric nanoparticles (NPs) shortly after ultrasound-guided delivery via the portal vein in early second-trimester fetal rhesus monkeys. Results demonstrated that poly(lactic-co-glycolic acid) (PLGA) NPs (N = 3; 3 mg administered at 75-80 days gestational age) and poly(amine-co-ester)-polyethylene glycol (PACE-PEG) NPs (N = 3; 3 mg at 75-80 days gestational age) distributed to fetal tissues when assessed 24 h post-administration. No adverse findings were observed. PLGA NPs were found primarily in the fetal liver and spleen, whereas PACE-PEG NPs showed more widespread biodistribution to a range of anatomical sites. In another fetal subset with PACE-PEG NPs (N = 2; 90 days gestational age) assessed within 48-h post-administration, results demonstrated enhanced green fluorescent protein reporter mRNA expression in select tissues. These early-stage short-term studies suggest that polymeric NPs, particularly those composed of PACE-PEG, can be safely administered and are potential candidates for fetal delivery of therapeutic nucleic acids. While preliminary, these studies provide evidence to support further investigations in this species to address long-term safety and efficiency.

Recently, our research group generated induced tissue-specific stem/progenitor (iTS/iTP) cells. Compared with induced pluripotent stem (iPS) cells, iTS/iTP cells offer several advantages, that is, easy generation, higher differentiation efficiency, and no teratoma formation. In this study, iTS cells were generated from mouse pancreatic tissues (iTS-P cells) using two different methods. Plasmid vectors were used for expressing Oct3/4, Sox2, Klf4, c-Myc (OSKM), or Yap1 (YAP) to evaluate the efficiency and differentiation potential of the resulting cells. No significant difference in reprogramming efficiency between the OSKM- and YAP-based methods was observed. Among the established clones, iTS-P OSKM2 and iTS-P YAP9 cells, which demonstrated high efficiency in differentiating to insulin-producing cells (IPCs), were selected for further comparison. Both iTS-P OSKM2 and iTS-P YAP9 cells expressed genetic markers of endoderm and pancreatic progenitors and differentiated into IPCs more efficiently than the embryonic stem (ES) cells. Genomic bisulfite sequencing revealed that the pluripotency factors Oct3/4 and Nanog were partially methylated in both iTS-P OSKM2 and iTS-P YAP9 cells. Unsupervised hierarchical clustering of gene expression profiles showed that iTS-P YAP9 cells clustered more closely with the ES cells than with the iTS-P OSKM2 cells. However, the expression levels of Oct3/4 and Nanog were significantly lower in both iTS-P OSKM2 and YAP9 cells than in the ES cells. These results conclude that no substantial difference is present in the characteristics between iTS-P cells induced by OSKM or YAP, and that their higher differentiation efficiency than the ES cells indicates promising potential for clinical applications.

Hearing impairment, one of the most prevalent sensory disorders, remains a major risk factor for dementia in the aging population. Although interventions such as hearing aids and cochlear implants provide partial benefit, they do not address the underlying pathology of sensorineural hearing loss. Inner ear gene therapy has attracted significant attention as a promising approach; however, its clinical translation requires minimally invasive and controllable methods for gene activation. We previously developed a photoactivatable Cre recombinase (PA-Cre) system for spatiotemporal regulation of gene expression. In this study, we evaluated the feasibility of irradiating the external auditory canal (EAC) and tympanic membrane (TM) as minimally invasive approaches for activating cochlear gene expression. Tyrosine-mutant AAV9/3 vectors (AAV.GTX) encoding PA-Cre and a Cre-dependent reporter (sfGFP-to-tdTomato) were injected via the round window membrane in 9-week-old C57BL/6J mice. Seven days later, light irradiation was applied using three approaches: (1) Direct cochlear irradiation via postauricular access, (2) TM irradiation with a fiber-optic probe, and (3) noninvasive EAC irradiation through the intact TM. Recombination efficiency in inner hair cells (IHCs) was quantified using whole-mount immunohistochemistry. AAV.GTX efficiently transduced IHCs and drove robust sfGFP expression. In the absence of light, tdTomato expression remained minimal (<5%), indicating low basal Cre leak activity. Direct cochlear irradiation produced strong recombination (conversion rate: 88.4 ± 1.5%), confirming the functionality of PA-Cre in the mouse inner ear. TM and EAC irradiation yielded high conversion efficiencies (95.8 ± 1.7% and 97.6 ± 1.2%, respectively), comparable to direct irradiation, while preserving cochlear integrity. These findings indicate that PA-Cre functions effectively in the mouse cochlea with minimal leak activity and that TM and EAC irradiation enable robust, minimally invasive gene activation. This strategy highlights the light-mediated, noninvasive modulation of cochlear gene expression, informing future translational development.