Human Gene Therapy

Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA has been documented for over two decades. Results from mouse and dog studies have raised concerns of insertional mutagenesis and clonal expansion following AAV exposure, particularly in the context of gene therapy. This study aimed to characterize the genomic location, abundance, and expansion of wtAAV and rAAV integrations in macaque and human genomes. Using an unbiased, next-generation sequencing-based approach, we identified the genome-wide integration loci in tissue samples (primarily liver) in 168 nonhuman primates (NHPs) and 85 humans naïve to rAAV exposure and 86 NHPs treated with rAAV in preclinical studies. Our results suggest that rAAV and wtAAV integrations exhibit similar, broad distribution patterns across species, with a higher frequency in genomic regions highly vulnerable to DNA damage or close to highly transcribed genes. rAAV exhibited a higher abundance of unique integration loci, whereas wtAAV integration loci were associated with greater clonal expansion. This expansive and detailed characterization of AAV integration in NHPs and humans provides key translational insights, with important implications for the safety of rAAV as a gene therapy vector.

Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60–90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

Asthma is a chronic inflammatory disease around the world. Extracellular adenosine triphosphate works as a dangerous signal in responding to cellular stress, irritation, or inflammation. It has also been reported its association with the pathogenicity in asthma, with increased level in lungs of asthmatics. Pannexin-1 is one of the routes that contributes to the release of adenosine triphosphate form intracellular to extracellular. The aim of this study was to apply pannexin-1 peptide antagonist ¹⁰Panx1 into adeno-associated viral (AAV) vectors on ovalbumin (OVA)-induced asthmatic mouse model. The results demonstrated that this treatment was able to reduce the adenosine triphosphate level in bronchoalveolar lavage fluid and downregulate the major relevant to the symptom of asthma attack, airway hyperresponsiveness to methacholine. The histological data also gave a positive support with decreased tissue remodeling and mucus deposition. Other asthmatic related features, including eosinophilic inflammation and OVA-specific T helper type 2 responses, were also decreased by the treatment. Beyond the index of inflammation, the proportion of effector and regulatory T cells was examined to survey the potential mechanism behind. The data provided a slightly downregulated pattern in lung GATA3⁺ CD4 T cells. However, an upregulated population of CD25⁺FoxP3⁺ CD4 T cells was seen in spleens. These data suggested that exogeneous expression of ¹⁰Panx1 peptide was potential to alleviated asthmatic airway inflammation, and this therapeutic effect might be from ¹⁰Panx1-mediated disruption of T cell activation or differentiation. Collectively, AAV vector-mediated ¹⁰Panx1 expression could be a naval therapy option to develop.

Gene therapy (GT) has emerged as a promising treatment option for disorders in the hematopoietic system, particularly primary immunodeficiencies (PID). Hematopoietic stem cells (HSCs) have gained attention due to their ability to support long-term hematopoiesis. In this study, we present a summary of research evaluating the most effective method of gene editing in HSCs for translational medicine. We conducted a systematic literature search in various databases, including Cochrane, LILACs, SciELO, and PubMed (MEDLINE), covering the period from January 1989 to June 10, 2023. The aim of this study was to identify articles that assessed the efficiency of gene editing in HSCs and clinical trials focusing on PID. Our research protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42022349850). Of the 470 studies identified in our search, 77 met the inclusion criteria. Among these, 61 studies were included in strategy 1 (gene therapy using HSC [GT-HSC]) of the systematic review (SR). We performed a meta-analysis on 17 of these studies. In addition, 16 studies were categorized under strategy 2 (clinical trials for PID). While clinical trials have demonstrated the potential benefits of GT-HSC, the safety and efficacy of gene editing still pose significant challenges. Various viral and nonviral approaches for gene delivery have been explored in basic and clinical research, with viral vectors being the most commonly used method in HSC therapeutics. Although promising, recent technologies such as CRISPR/Cas are not yet ready for efficient long-term restoration of the immune system as a whole.

Cystic fibrosis (CF) is potentially treatable by gene therapy. Since the identification of the CF gene, preclinical and clinical trials have concentrated on achieving effective gene therapy targeting the lung. However, the lung has proven to be a formidable barrier to successful gene therapy especially for CF, and many clinical trials failed to achieve efficacy. Recent advances in vector design and adeno-associated virus (AAV) serotypes have increased the chances of success. Given that CF is a multi-organ disease, the goal of this study was to test whether a gene therapy approach involving AAV1 or AAV6 vector delivery via the systemic circulation would at the same time overcome the barrier of lung delivery and transduce organs commonly affected by CF. To accomplish this, we sprayed AAV1 containing green fluorescent protein (GFP) into the trachea or injected it intravenously (IV). We also tested AAV6 injected IV. No adverse events were noted. Ferrets were necropsied 30 days after vector delivery. AAV1 or AAV6 vector genomes, messenger RNA (mRNA) expression, and GFP were detected in all the tracheal and lung samples from the treated animals, whether AAV1 was sprayed into the trachea or injected IV or AAV6 was injected IV. Importantly, both surface epithelial and basal cells of the trachea and lung airways were successfully transduced, regardless of which route of delivery or vector serotype used for transduction. We detected also AAV1 and AAV6 vector genomes, mRNA expression, and GFP in the livers and pancreases, particularly in the acinar cells of the pancreatic duct. These data suggest that gene transfer is attainable in the airways, liver, and pancreas using either serotype, AAV1 or AAV6. Given that these same organs are affected in CF, systemic delivery of AAV may be the preferred route of delivery for a gene therapy for CF.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation of CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 single-chain fragment variable region (scFv), as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality. We have since improved the platform to create a superior cellular product—CAR T cells secreting single-chain trimeric 4-1BB ligand fused to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocyte proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder antitumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CD28-costimulated CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand. In vitro and in vivo, CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy of αPD1-41BBL-secreting CAR T cells.

Lentiviral (LV) vector-based gene therapy is gaining popularity for treating a wide range of diseases. Various LV vectors are being developed for transducing cells in cellular gene therapy at St. Jude. Some LV vectors are produced using stable 293T packaging cell lines, which includes gag-pol-rev-tat and virus-glycoprotein. Transactivating factor (transactivator of transcription [Tat]) is a regulatory protein that drastically increases the efficiency of lentiviral transcription. Residual analysis of Tat is critical for gene vector quality and safety. In this work, we developed a highly sensitive liquid chromatography-tandem mass spectrometry method for analysis of residual Tat in Lentivirus as an alternative to enzyme-linked immunosorbent assay. Residual Tat in LV can be accurately quantified with high specificity with a limit of detection of 0.3 ng/mL.