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Long-term follow-up of 12 persons with Leber's congenital amaurosis treated with gene therapy showed that about half of them had improvements in retinal sensitivity (although the extent varied markedly among patients), followed by a decline. Leber’s congenital amaurosis is a group of inherited, early-onset, severe retinal dystrophies that cause substantial sight impairment in childhood. 1 One of the causes of this condition is mutations in the gene encoding RPE65 (retinal pigment epithelium–specific protein 65 kDa). The encoded retinoid isomerase converts all- trans retinyl esters to 11- cis retinal for the regeneration of visual pigment after exposure to light. RPE65 deficiency causes photoreceptor-cell dysfunction and impaired vision from birth. Severe dysfunction of rod photoreceptor cells, which are wholly reliant on retinal pigment epithelium–derived RPE65, causes severely impaired night vision. The function of cone photoreceptor cells, which mediate . . .

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

Gene transfer is an attractive option to treat the basic defect in cystic fibrosis. In a double-blind, placebo-controlled, rising-dose tolerance study in the nasal epithelium, we tested the safety and efficacy of a cationic liposome [p-ethyl-dimyristoylphosphadityl choline (EDMPC) cholesterol] complexed with an expression plasmid containing hCFTR cDNA. Eleven adult CF patients were studied in a protocol that allowed comparisons within individual subjects: vector and placebo were sprayed into alternate nostrils at intervals over 7 h. After dosing, vector-specific DNA was present in nasal lavage of all subjects for up to 10 days. There were no adverse events. The vector-treated epithelium did not exhibit a significant increase in CFTR-mediated Cl− conductance from baseline and was not different from the placebo-treated nostril: mean ΔCFTR Cl− conductance, mV ± SEM, −1.6 ± 0.4 vs −0.6 ± 0.4, respectively. CFTR-mediated Cl− conductance increased toward normal during repetitive nasal potential difference measurements over the 3 days before dosing which influenced the postdosing calculations. No vector-specific mRNA was detected in the nasal epithelial scrape biopsies, although endogenous CFTR mRNA was detected in all subjects. We conclude that the lipid–DNA complex is safe, but did not produce consistent evidence of gene transfer to the nasal epithelium by physiologic or molecular measures.

Genetic deficiency of otoferlin, a protein critical to synaptic transmission by the sensory hair cells of the ear, causes congenital deafness. Medicines to treat the condition are lacking; children typically receive cochlear implants. DB-OTO is a dual adeno-associated virus 1 gene therapy that delivers human complementary DNA (encoding otoferlin) regulated by a hair cell-specific promoter. We conducted an open-label, single-group, first-in-human registrational study to evaluate DB-OTO. Children with variants and profound deafness (defined by an average audiometric threshold of >90 decibel hearing level [dB HL], indicating an inability to hear a gas-powered lawn mower) received an intracochlear infusion of DB-OTO (7.2×10 vector genomes per ear) in one or both ears. The primary efficacy end point was an average threshold on behavioral pure-tone audiometry (PTA) at week 24 of 70 dB HL or less, a clinical standard that generally avoids cochlear implantation and enables natural acoustic hearing. A key secondary end point was the presence of an auditory brain-stem response to a click stimulus at a threshold at or below 90 dB normalized hearing level (db nHL) at week 24. Safety assessments included adverse events, laboratory results, and vestibular testing. A total of 12 children have been enrolled in the study. After a single infusion of DB-OTO, a PTA average threshold of 70 dB HL or less at week 24 (primary end point) and an auditory brain-stem response at or below 90 dB nHL (key secondary end point) were found in 9 of the 12 participants (75%; 95% confidence interval, 43 to 95; P = 1.1×10 for both end points). Six participants could hear soft speech without assistive devices, and 3 had average normal hearing sensitivity. A total of 67 adverse events occurred or worsened during or after treatment, none of which led to discontinued participation in the study. DB-OTO gene therapy improved hearing in patients with -related deafness, enabling natural acoustic hearing and normalizing hearing sensitivity in 3 of 12 treated patients. (Funded by Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT05788536.).

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.

Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator ( CFTR ) complementary DNA were administered to the sheep lung ( n =8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1–10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.

Trichinellosis has major economic impacts on animal husbandry and food safety, and the control and elimination of trichinellosis is a major objective of veterinary medicine. A gene encoding serine protease of Trichinella spiralis (Ts-Adsp) was identified by immunoscreening an adult T. spiralis cDNA library. In this study, the recombinant Ts-Adsp protein (rTs-Adsp) was cloned and expressed in a prokaryotic expression system and purified by Ni-affinity chromatography. To determine whether the purified rTs-Adsp is a potential vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with this protein in combination with an alum adjuvant and subsequently challenged with T. spiralis larvae. The results showed that mice vaccinated with rTs-Adsp exhibited an average reduction in the muscle larvae burden of 46.5% relative to the control group. Immunization with the rTs-Adsp antigen induced both humoral and cellular immune responses, which manifested as elevated specific anti–rTs-Adsp IgG and IgE antibodies and a mixed Th1–Th2 response, as determined by Th1 (IFN-γ and IL-2) and Th2 (IL-4, IL-10, and IL-13) cytokine profiling, with the Th2 predominant. Thus, purified rTs-Adsp is able to limit the invasion of T. spiralis, and this protein could be an effective vaccine candidate for trichinellosis.

Blood-brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and D-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF.