Gene Therapy in Patients with the Crigler-Najjar Syndrome.

BACKGROUND: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 µmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 µmol per liter. The patients who received the higher dose had bilirubin levels below 300 µmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 µmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 µmol per liter). CONCLUSIONS: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B.

BACKGROUND: Moderate-to-severe hemophilia B is treated with lifelong, continuous coagulation factor IX replacement to prevent bleeding. Gene therapy for hemophilia B aims to establish sustained factor IX activity, thereby protecting against bleeding without burdensome factor IX replacement. METHODS: In this open-label, phase 3 study, after a lead-in period (≥6 months) of factor IX prophylaxis, we administered one infusion of adeno-associated virus 5 (AAV5) vector expressing the Padua factor IX variant (etranacogene dezaparvovec; 2×1013 genome copies per kilogram of body weight) to 54 men with hemophilia B (factor IX activity ≤2% of the normal value) regardless of preexisting AAV5 neutralizing antibodies. The primary end point was the annualized bleeding rate, evaluated in a noninferiority analysis comparing the rate during months 7 through 18 after etranacogene dezaparvovec treatment with the rate during the lead-in period. Noninferiority of etranacogene dezaparvovec was defined as an upper limit of the two-sided 95% Wald confidence interval of the annualized bleeding rate ratio that was less than the noninferiority margin of 1.8. Superiority, additional efficacy measures, and safety were also assessed. RESULTS: The annualized bleeding rate decreased from 4.19 (95% confidence interval [CI], 3.22 to 5.45) during the lead-in period to 1.51 (95% CI, 0.81 to 2.82) during months 7 through 18 after treatment, for a rate ratio of 0.36 (95% Wald CI, 0.20 to 0.64; P<0.001), demonstrating noninferiority and superiority of etranacogene dezaparvovec as compared with factor IX prophylaxis. Factor IX activity had increased from baseline by a least-squares mean of 36.2 percentage points (95% CI, 31.4 to 41.0) at 6 months and 34.3 percentage points (95% CI, 29.5 to 39.1) at 18 months after treatment, and usage of factor IX concentrate decreased by a mean of 248,825 IU per year per participant in the post-treatment period (P<0.001 for all three comparisons). Benefits and safety were observed in participants with predose AAV5 neutralizing antibody titers of less than 700. No treatment-related serious adverse events occurred. CONCLUSIONS: Etranacogene dezaparvovec gene therapy was superior to prophylactic factor IX with respect to the annualized bleeding rate, and it had a favorable safety profile. (Funded by uniQure and CSL Behring; HOPE-B ClinicalTrials.gov number, NCT03569891.).

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue.

Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.

Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

Gene therapy of prostate cancer using liposomes containing perforin expression vector driven by the promoter of prostate-specific antigen gene.

Perforin secreted from cytotoxic lymphocytes plays a critical role in cancer immunosurveillance. The aim of this study was to investigate the therapeutic potential of liposomes containing perforin expression vector driven by the promotor of prostate-specific antigen (PSA). The anti-tumor effect of perforin was analyzed using prostate cancer (PC) PC-3 cells in which perforin expression was controlled by Tet-on system (PC-3PRF cells). Liposomes encapsulating PSA promoter-driven perforin expression vector (pLipo) were constructed for its specific expression in PC. The anti-tumor effect of pLipo was evaluated in vitro using docetaxel-resistant PC 22Rv1 PC cell line, 22Rv1DR, and PC-3 cells in the presence of human peripheral blood mono nuclear cells (PBMCs) and also in vivo using male nude mice bearing 22Rv1DR cell-derived tumor xenograft. Induction of perforin significantly inhibited growth of PC-3PRF cells. Treatment with pLipo induced perforin expression in 22Rv1DR cells expressing PSA but not in PC-3 cells lacking it. Treatment with pLipo at a low concentration was prone to inhibit growth of both cell lines and significantly inhibited growth of 22Rv1DR cells when co-incubated with PBMCs. The combined use of pLipo at a high concentration with PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. Intravenous administration of pLipo via tail vein increased the level of perforin in tumor and serum and significantly decreased the tumor volume. Our results suggest that liposome-mediated PC-specific expression of perforin could be a novel therapy for advanced PC.

Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes.

Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

Calcium Imaging Reveals Fast Tuning Dynamics of Hippocampal Place Cells and CA1 Population Activity during Free Exploration Task in Mice.

Hippocampal place cells are a well-known object in neuroscience, but their place field formation in the first moments of navigating in a novel environment remains an ill-defined process. To address these dynamics, we performed in vivo imaging of neuronal activity in the CA1 field of the mouse hippocampus using genetically encoded green calcium indicators, including the novel NCaMP7 and FGCaMP7, designed specifically for in vivo calcium imaging. Mice were injected with a viral vector encoding calcium sensor, head-mounted with an NVista HD miniscope, and allowed to explore a completely novel environment (circular track surrounded by visual cues) without any reinforcement stimuli, in order to avoid potential interference from reward-related behavior. First, we calculated the average time required for each CA1 cell to acquire its place field. We found that 25% of CA1 place fields were formed at the first arrival in the corresponding place, while the average tuning latency for all place fields in a novel environment equaled 247 s. After 24 h, when the environment was familiar to the animals, place fields formed faster, independent of retention of cognitive maps during this session. No cumulation of selectivity score was observed between these two sessions. Using dimensionality reduction, we demonstrated that the population activity of rapidly tuned CA1 place cells allowed the reconstruction of the geometry of the navigated circular maze; the distribution of reconstruction error between the mice was consistent with the distribution of the average place field selectivity score in them. Our data thus show that neuronal activity recorded with genetically encoded calcium sensors revealed fast behavior-dependent plasticity in the mouse hippocampus, resulting in the rapid formation of place fields and population activity that allowed the reconstruction of the geometry of the navigated maze.

Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety.

Schwannomas are tumors derived from Schwann-lineage cells, cells that protect and support myelinated nerves in the peripheral nervous system. They are typically slow-growing, encapsulated and benign. These tumors develop along peripheral, spinal and cranial nerves causing pain, sensory-motor dysfunction and death. Primary treatment for schwannoma is operative resection which can be associated with significant morbidity. Pharmacotherapy is largely restricted to bevacizumab, which has minimal or no efficacy for many patients and can be associated with treatment-limiting adverse effects. Given the suffering and morbidity associated with schwannoma and the paucity of therapeutic options, there is an urgent need for safe and effective therapies for schwannomas. We previously demonstrated that adeno-associated virus serotype 1 (AAV1) vector mediated delivery of the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) under the control of the P0 promoter, produced a prolonged reduction in tumor volume and tumor-associated pain in human xenograft and mouse syngeneic schwannoma models. Here, we present data essential for the translation of our AAV1-P0-ASC schwannoma gene therapy to clinical trials. We determine the minimum effective dose of AAV1-P0-hASC required to induce an anti-tumor effect in the xenograft human-schwannoma model. We also show that the presence of preexisting AAV1 immunity does not alter the antitumor efficacy of AAV-P0-mASC in a syngeneic mouse schwannoma model. Furthermore, the maximum deliverable intratumoral dose of AAV1-P0-ASC was not associated with neuronal toxicity in immunocompetent mice. Taken together, these safety and efficacy data support the translation of the AAV1-P0-ASC schwannoma gene therapy strategy to clinical trials.

Efficacy of mRNA, adenoviral vector, and perfusion protein COVID-19 vaccines.

Coronavirus disease 2019 (COVID-19) has a devastating impact on global populations triggered by a highly infectious viral sickness, produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The third major cause of mortality in the United States, following heart disease and cancer in 2020, was undoubtedly COVID-19. The centers for disease control and prevention (CDC) and the world health organization (WHO) separately developed a categorization system for differentiating new strains of SARS-CoV-2 into variants of concern (VoCs) and variants of interest (VoIs) with the continuing development of various strains SARS-CoV-2. By December 2021, five of the SARS-CoV-2 VoCs were discovered from the onset of the pandemic depending on the latest epidemiologic report by the WHO: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Mutations in the receptor-binding domain (RBD) and n-terminal domain (NTD) have been found throughout all five identified VoCs. All strains other than the delta mutant are often found with the N501Y mutation situated on the RBD, resulting in higher binding between the spike protein and angiotensin-converting enzyme 2 (ACE2) receptors, enhanced viral adhesion, and following the entrance to host cells. The introduction of these new strains of SRAS-CoV-2 is likely to overcome the remarkable achievements gained in restricting this viral disease to the point where it is presented with remarkable vaccine developments against COVID-19 and strong worldwide mass immunization initiatives. Throughout this literature review, the effectiveness of current COVID-19 vaccines for managing and prohibiting SARS-CoV-2 strains is thoroughly described.

Peripherally delivered Adeno-associated viral vectors for spinal cord injury repair.

Via the peripheral and autonomic nervous systems, the spinal cord directly or indirectly connects reciprocally with many body systems (muscular, intengumentary, respiratory, immune, digestive, excretory, reproductive, cardiovascular, etc). Accordingly, spinal cord injury (SCI) can result in catastrophe for multiple body systems including muscle paralysis affecting movement and loss of normal sensation, as well as neuropathic pain, spasticity, reduced fertility and autonomic dysreflexia. Treatments and cure for an injured spinal cord will likely require access of therapeutic agents across the blood-CNS (central nervous system) barrier. However, some types of repair within the CNS may be possible by targeting treatment to peripherally located cells or by delivering Adeno-Associated Viral vectors (AAVs) by peripheral routes (e.g., intrathecal, intravenous). This review will consider some future possibilities for SCI repair generated by therapeutic peripheral gene delivery. There are now six gene therapies approved worldwide as safe and effective medicines of which three were created by modification of the apparently nonpathogenic Adeno-Associated Virus. One of these AAVs, Zolgensma, is injected intrathecally for treatment of spinal muscular atrophy in children. One day, delivery of AAVs into peripheral tissues might improve recovery after spinal cord injury in humans; we discuss experiments by us and others delivering transgenes into nerves or muscles for sensorimotor recovery in animal models of SCI or of stroke including human Neurotrophin-3. We also describe ongoing efforts to develop AAVs that are delivered to particular targets within and without the CNS after peripheral administration using capsids with improved tropisms, promoters that are selective for particular cell types, and methods for controlling the dose and duration of expression of a transgene. In conclusion, in the future, minimally invasive administration of AAVs may improve recovery after SCI with minimal side effects.

Chemically modified mRNA beyond COVID-19: Potential preventive and therapeutic applications for targeting chronic diseases.

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.

ß-arrestin-2 alleviates rheumatoid arthritis injury by suppressing NLRP3 inflammasome activation and NF- κB pathway in macrophages.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder that inflicts damage to the joints of the hands and wrist. The aim of this study was to investigate the protective effect of ß-Arrestin-2 (ßArr2) on RA in vivo and in vitro. The ßArr2 adenovirus (ßArr2-Ad) or the control (Con-Ad) was injected into the ankle joint cavity of collagen-induced arthritis (CIA) mice. According to the results, an improvement was shown in the symptoms and pathological injury of RA after an upregulation of ßArr2. Correspondingly, the inflammatory response was attenuated, as evidenced by the decreased serum pro-inflammatory cytokines levels and NF-κB pathway-related proteins. Nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome activation was inhibited in CIA mice treated with ßArr2-Ad injection, as reflected by the diminished IL-18 level and declined protein levels of inflammasome components in the ankle joint. Likewise, the anti-inflammatory effect of macrophages was also validated by in vitro experiments. In summary, ßArr2 effectively ameliorates ankle inflammation in CIA mice via NF-κB/NLRP3 inflammasome, providing theoretical and clinical basis for RA therapy.

Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors.

Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Risk Mitigation of Immunogenicity: A Key to Personalized Retinal Gene Therapy.

Gene therapy (GT) for ocular disorders has advanced the most among adeno-associated virus (AAV)-mediated therapies, with one product already approved in the market. The bank of retinal gene mutations carefully compiled over 30 years, the small retinal surface that does not require high clinical vector stocks, and the relatively immune-privileged environment of the eye explain such success. However, adverse effects due to AAV-delivery, though rare in the retina have led to the interruption of clinical trials. Risk mitigation, as the key to safe and efficient GT, has become the focus of 'bedside-back-to-bench' studies. Herein, we overview the inflammatory adverse events described in retinal GT trials and analyze which components of the retinal immunological environment might be the most involved in these immune responses, with a focus on the innate immune system composed of microglial surveillance. We consider the factors that can influence inflammation in the retina after GT such as viral sensors in the retinal tissue and CpG content in promoters or transgene sequences. Finally, we consider options to reduce the immunological risk, including dose, modified capsids or exclusion criteria for clinical trials. A better understanding and mitigation of immune risk factors inducing host immunity in AAV-mediated retinal GT is the key to achieving safe and efficient GT.

Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery.

To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.

Protocol for cortical-wide field-of-view two-photon imaging with quick neonatal adeno-associated virus injection.

We recently established a simple and versatile adeno-associated virus (AAV) induction approach that enables dense (>90% labeled neurons) and cortical-wide Ca2+ sensor expression. Here, we describe the stepwise protocol for neonatal AAV injection of a Ca2+ sensor. We also detail the steps for subsequent craniotomy to generate a chronic cranial window, followed by wide-field two-photon Ca2+ imaging in an awake mouse. This protocol serves as an alternative to the use of transgenic animals and offers translatable options for cortical-wide experiments. For complete details on the use and execution of this protocol, please refer to Ota et al. (2021).

Evaluation of the Central Effects of Systemic Lentiviral-Mediated Leptin Delivery in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes (T1D) is characterized by hyperphagia, hyperglycemia and activation of the hypothalamic-pituitary-adrenal (HPA) axis. We have reported previously that daily leptin injections help to alleviate these symptoms. Therefore, we hypothesized that leptin gene therapy could help to normalize the neuroendocrine dysfunction seen in T1D. Adult male Sprague Dawley rats were injected i.v. with a lentiviral vector containing the leptin gene or green fluorescent protein. Ten days later, they were injected with the vehicle or streptozotocin (STZ). HPA function was assessed by measuring norepinephrine (NE) levels in the paraventricular nucleus (PVN) and serum corticosterone (CS). Treatment with the leptin lentiviral vector (Lepvv) increased leptin and insulin levels in non-diabetic rats, but not in diabetic animals. There was a significant reduction in blood glucose levels in diabetic rats due to Lepvv treatment. Both NE levels in the PVN and serum CS were reduced in diabetic rats treated with Lepvv. Results from this study provide evidence that leptin gene therapy in STZ-induced diabetic rats was able to partially normalize some of the neuroendocrine abnormalities, but studies with higher doses of the Lepvv are needed to develop this into a viable option for treating T1D.