Replication-deficient adenovirus-based gene therapy vectors were the first vectors demonstrated to mediate effective, robust in vivo gene transfer. The ease of genome engineering, large carrying capacity, and methods for large-scale vector production made adenoviral vectors a primary focus in the early days of gene therapy. Many vector modifications such as capsid engineering and regulated and cell-specific transgene expression were first demonstrated in adenovirus (Ad) vectors. However, early human studies proved disappointing, with safety and efficacy issues arising from anti-vector innate and acquired immune responses. While many gene therapy researchers moved to other vectors, others recognized that the immune response and limited duration of transgene expression were useful in the correct context. The striking example of this was the use of several effective adenovirus vectors engineered as COVID-19 vaccines estimated to have been administered to 2 billion people. In addition to vaccines, current applications of Ad vectors relate to anti-cancer therapies, tissue remodeling, and gene editing.

Abstract Spastic paraplegia 47 (SPG47) is a neurological disorder caused by mutations in the adaptor protein complex 4 β1 subunit ( AP4B1 ) gene leading to AP-4 complex deficiency. SPG47 is characterised by progressive spastic paraplegia, global developmental delay, intellectual disability and epilepsy. Gene therapy aimed at restoring functional AP4B1 protein levels is a rational therapeutic strategy to ameliorate the disease phenotype. Here we report that a single delivery of adeno-associated virus serotype 9 expressing hAP4B1 (AAV9/hAP4B1) into the cisterna magna leads to widespread gene transfer and restoration of various hallmarks of disease, including AP-4 cargo (ATG9A) mislocalisation, calbindin-positive spheroids in the deep cerebellar nuclei, anatomical brain defects and motor dysfunction, in an SPG47 mouse model. Furthermore, AAV9/hAP4B1-based gene therapy demonstrated a restoration of plasma neurofilament light (NfL) levels of treated mice. Encouraged by these preclinical proof-of-concept data, we conducted IND-enabling studies, including immunogenicity and GLP non-human primate (NHP) toxicology studies. Importantly, NHP safety and biodistribution study revealed no significant adverse events associated with the therapeutic intervention. These findings provide evidence of both therapeutic efficacy and safety, establishing a robust basis for the pursuit of an IND application for clinical trials targeting SPG47 patients.

Chronic hypereosinophilia, defined as persistent elevated blood levels of eosinophils ≥1,500/μL, is associated with tissue infiltration of eosinophils and consequent organ damage by eosinophil release of toxic mediators. The current therapies for chronic hypereosinophilia have limited success, require repetitive administration, and are associated with a variety of adverse effects. As a novel approach to treat chronic hypereosinophilia, we hypothesized that adeno-associated virus (AAV)-mediated delivery of an anti-human eosinophil antibody would provide one-time therapy that would mediate persistent suppression of blood eosinophil levels. To assess this hypothesis, we first generated a human monoclonal antibody (mAb) directed against Siglec8, a sialic-acid binding immunoglobulin-like lectin, expressed at high levels on the cell surface of human eosinophils. Transgenic mice with a human immunoglobulin repertoire were immunized with human Siglec8 protein or DNA encoding human Siglec8. Based on target binding assessments, the 08C07 mAb was chosen for further study. The human variable regions of 08C07 were joined to the human Ig constant region, creating H08C07 (hAntiEos), a fully human anti-human eosinophil mAb. Using the gene sequence of hAntiEos, we created AAVrh.10hAntiEos, an AAVrh.10-based vector expressing the heavy and light chains of H08C07. Intravenous administration of AAVrh.10hAntiEos (1011 genome copies or gc) to C57Bl/6 mice resulted in persistent elevated serum levels of hAntiEos. In vivo gene therapy generated hAntiEos bound to recombinant human Siglec8 protein in a dose-dependent manner and to human eosinophils, mediated apoptosis of human eosinophils, and antibody-dependent cellular cytotoxicity activity against human eosinophils. Consistent with these data, administration of AAVrh.10hAntiEos to human CD34+ transplanted NSG-SGM3 immunodeficient mice suppressed levels of human eosinophils in vivo. AAVrh.10hAntiEos holds the potential to offer therapeutic benefit to patients with chronic hypereosinophilia.

Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials.

Supplementary Methods and Materials, Supplementary Tables 1-2, Supplemental Figure Legends, Supplementary Figure 1 from Modulation of Cystatin A Expression in Human Airway Epithelium Related to Genotype, Smoking, COPD, and Lung Cancer

PURPOSE: Autologous fat transfer is an increasingly common aesthetic and reconstructive technique used for facial rejuvenation, breast or buttock contouring and augmentation, and improving the form and function of atrophic conditions like scleroderma, scar contractures and radiation. The long-term results are often disappointing because of variable and often significant loss of up to 70% or more of the graft. Other complications associated with this procedure are fat necrosis/nodule formation and the development of oil cysts. A major cause of graft loss and its associated complications is inadequate vascularization of the fat following transplantation. Thus far attempts to incorporate various growth factors to augment graft take have not been successful likely because of inadequate bioavailability over the initial 24-72 hours after transplant. Thus, we propose to solve this challenge by genetically modifying harvested fat prior to transplantation with an adenovirus gene transfer vector coding for all 3 major isoforms of vascular endothelial growth factor (capable of inducing expression for 1-2 weeks), to improve angiogenesis and promote engraftment of transplanted fat. METHODS: Fat was harvested using standard liposuction techniques from several patient donors and concentrated by Telfa rolling to remove extra lipid and fluid. Ad5VEGF-All6A+ is a E1‾E3‾ serotype 5 adenovirus (Ad) vector coding for a novel genomic hybrid coding for all 3 major isoforms of human vascular endothelial growth factor (VEGF 121, 165 and 189). The vector was produced and stored at - 70C. At the time of vector delivery, the vector was thawed, diluted, and administrated to the harvested autologous fat at different concentrations (109, 1010 and 1011 pfu). The expression of VEGF was assessed in triplicate by quantitative PCR (RNA) and by ELISA (protein) using cell lysate and media supernatant following a 24-hour incubation. Untouched fat, PBS treated, and Ad-null (1011 pfu) administration served as controls. RESULTS: The highest RNA expression of VEGFA, measured by qPCR, was achieved by the 109pfu dose of Ad5VEGF-All6A+. Absolute mRNA VEGFA copy numbers were 6.70E+06 (±3.65E+05), 4.21E+06 (2.97 E+05) and 2.59E+06 (3.19 E+05), respectively for each increasing dose. VEGFA protein data exhibited a dose dependent response for each of the increasing Ad5VEGF-All6A+ doses. VEGFA ELISA data measured in protein cell lysates and media supernatant showed an increasing amount of VEGFA produced. CONCLUSIONS: We have demonstrated the administration of Ad5VEGF-All6A+ to human lipoaspirate significantly increases VEGF expression. Importantly, the expression induced with this vector lasts between 1-2 weeks, which is crucial to maintain the angiogenic response necessary to rescue ischemic transplanted fat. This strategy has significant potential to significantly improve the vascularization of transplanted fat. Ongoing studies include in vivo animal fat grafting experiments to determine long term fat graft retention.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh. 10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig) G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-gamma in CD8(+) and interleukin (IL)-12 in CD4(+)], Th2 (IL-4, IL-5 and IL-13 in CD4(+)) and Th17 (IL-17 in CD4(+)). Interestingly, AdC7OprF. RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF. RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity.

The glutathione S-transferase (GST) enzymes catalyse the conjugation of xenobiotics to glutathione. Based on reports that inherited copy number variations (CNVs) modulate some GST gene expression levels, and that the small airway epithelium (SAE) and alveolar macrophages (AMs) are involved early in the pathogenesis of smoking-induced lung disease, we asked: do germline CNVs modulate GST expression levels in SAE and AMs? Microarrays were used to survey GST gene expression and determine CNVs genotypes in SAE and AMs obtained by bronchoscopy from current smokers and nonsmokers. 26% of subjects were null for both GSTM1 alleles, with reduced GSTM1 mRNA levels seen in both SAE and AMs. 30% of subjects had homozygous deletions of GSTT1, with reduced mRNA levels in both tissues. Interestingly, GSTT2B exhibited homozygous deletion in the blood of 27% of subjects and was not expressed in SAE in the remainder of subjects, but was expressed in AMs of heterozygotes and wild-type subjects, proportionate to genotype. These data show a germline CNV-mediated linear relationship of genotype with expression level, suggesting minimal compensation of gene expression levels in heterozygotes, consistent with GST polymorphisms playing a role in the risk of smoking-associated, xenobiotic-induced lung disease.