Adoptive Cell Therapies based on cytokine-induced killer cells (CIKs) can address the heterogeneity of solid tumors due to their multiple mechanisms of cancer cell recognition. However, tumor trafficking is one of the ma...Adoptive Cell Therapies based on cytokine-induced killer cells (CIKs) can address the heterogeneity of solid tumors due to their multiple mechanisms of cancer cell recognition. However, tumor trafficking is one of the main limitations. In this work, we describe that a high proportion of CIKs obtained from pancreatic ductal adenocarcinoma patients express the CXCR3 and CCR5 receptors, and they migrate towards their corresponding chemokines CXCL10 and CCL5 in vitro. Using an immune competent orthotopic PDAC mouse model, we have investigated the ability of different clinically compatible interventions to increase the expression of these chemokines. No significant elevation was obtained with chemotherapy (5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, gemcitabine or temozolomide), tyrosine kinase inhibitors sorafenib and sunitinib, or the immunostimulatory agents polyinosinic:polycytidylic acid, Mycobacterium tuberculosis antigens and diphtheria/pertussis/tetanus vaccine. In contrast, CXCL10 and CCL5 expression was stimulated by local administration of an adenoviral vector equipped with a drug-inducible expression system for interleukin-12 (IL-12). Combination of the vector and CIKs obtained a strong antitumor effect in the PDAC model, although it was mainly due to vector-mediated recruitment of endogenous immune cells. We conclude that additional barriers beyond chemokine expression should be overcome in order to unleash the full potential of CIKs on solid tumors.
Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced c...Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B186 and muSEAP, separated from PD-L1/2 by a self-cleaving P2A peptide. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L1/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice. Vectors (rAAV6-muSEAP, rAAV6-muSEAP-PD-L1, or rAAV6-muSEAP-PD-L2) were injected into two sites of the gastrocnemius muscle at a total dose of 1×10 vg. Co-delivery of PD-L1, particularly, significantly enhanced muSEAP secretion into the bloodstream up to 12 weeks despite elevated anti-AAV6 responses in pre-immunized mice. muSEAP secretion increased 33.3- and 31.4-fold with the co-delivery of PD-L1, while the increase was only 5.6- and 9.3-fold in the muSEAP control group at 5 and 12 weeks, respectively. Ligand-treated pre-immunized animals also had less T-cell infiltration into the treated muscle compared to naïve animals. In summary, co-delivery of PD-L1/2 alongside a transgene represents a promising strategy for achieving sustained gene expression in individuals pre-exposed to AAV.
Adeno-associated virus (AAV)-based gene therapies have garnered significant attention and investment due to their clinical success and potential to address underlying causes of many diseases. AAV vectors provide effectiv...Adeno-associated virus (AAV)-based gene therapies have garnered significant attention and investment due to their clinical success and potential to address underlying causes of many diseases. AAV vectors provide effective delivery of therapeutic genetic material to disease-relevant tissues. When evaluating safety and efficacy of recombinant AAV vectors, biodistribution profiles play a critical role in novel therapy development. Herein, a biodistribution metadata analysis was performed on ten studies involving 51 cynomolgus macaques (Macaca fascicularis). The macaques received a self-complementary or single-stranded AAV9 vector containing chicken ß-actin (CBA) or cytomegalovirus (CMV173) promoters expressing fluorescent reporters or a human SMN1 gene. These studies covered various routes of administration (ROA) including intravenous (IV), intracisternal magna (ICM), and lumbar puncture intrathecal (IT) injection. Metadata analysis of AAV9 biodistribution showed relatively uniform vector genome delivery throughout spinal cord tissues over multiple timepoints and ROAs. Moreover, decreased expression efficiency of viral DNA in liver was observed regardless of the ROA, macaque age, or viral construct used. To understand this trend, epigenetic profiling of tissue-localized AAV9 vector genome DNA was performed. Experimental evidence supports partial silencing and repression of transgene expression in macaque liver. These findings point to plausible strategies to consider in preclinical development of AAV9 mediated gene therapies.
This article provides a regulatory perspective on secondary malignancy of T-cell origin as a rare adverse reaction to the currently marketed CD19- or BCMA-directed chimeric antigen receptor (CAR) T-cell therapies. To ass...This article provides a regulatory perspective on secondary malignancy of T-cell origin as a rare adverse reaction to the currently marketed CD19- or BCMA-directed chimeric antigen receptor (CAR) T-cell therapies. To assess the risk, causality between reported suspected adverse reactions and CAR T-cell therapy was assessed applying the principles of the World Health Organization-Uppsala Monitoring Centre causality categories, alongside a review of scientific publications and data from registries/ databases. By 11 April 2024, 38 cases of T-cell malignancy after CAR T-cell therapy were reported in patients aged 29-80 years. In 19 patients, tumour samples were tested for the presence of CAR transgene, which was detected in seven cases. Most of the T-cell malignancies were diagnosed within 12 months of treatment (22/33; 67%). The reporting rate is approximately one case per 1000 patients treated. An overall causal relationship was established with at least a reasonable possibility. Regulatory measures included updates to the product information, risk management plan, and educational materials. An additional pharmacovigilance activity was requested from the marketing authorisation holders (MAHs) to strengthen the process of genetic testing of residual tumour samples. To further characterise this risk and understand underlying mechanisms, continued efforts from healthcare professionals, MAHs and regulators are essential. Well-documented case reports, including information on genetic testing of tumour samples, are considered crucial elements.
Retinitis pigmentosa (RP) associated with mutations in the rhodopsin gene (RHO) is a significant cause of blindness. Here we report on the application of adenine base editing of the c.1030C>T (p.Q344X) RHO mutation linke...Retinitis pigmentosa (RP) associated with mutations in the rhodopsin gene (RHO) is a significant cause of blindness. Here we report on the application of adenine base editing of the c.1030C>T (p.Q344X) RHO mutation linked to RP. Using a fluorescence reporter cell system, we optimized editing by exploring base editors, sgRNA, and delivery methods. Flow cytometry, western blotting, and immunofluorescence microscopy confirmed the restoration of full-length rhodopsin after editing. DNA sequencing verified editing at the target nucleotide and the absence of bystander edits within the editing window. Polyethylenimine cationic polymer transfection of cells with a plasmid containing the NG-ABE8e adenine base editor and A6 guide RNA that placed the targeted adenine in position 6 of the editing window resulted in 31.0% gDNA sequence correction and 26.3% rhodopsin protein correction as determined by flow cytometry. Purified NG-ABE8e protein complexed with A6-sgRNA showed 32.2% gDNA editing and 44.5% rhodopsin correction. Plasmid NG-ABE8e and A6-sgRNA co-encapsulated into lipid nanoparticles (LNPs) and transfected into the reporter cell system resulted in the highest editing (42.6% gDNA editing and 65.9% rhodopsin correction). These results demonstrate the successful correction of the c.1030C>T RHO mutation and provide the foundation for base editing as a treatment for RP.
Adeno-associated viruses (AAVs) hold significant promise for gene therapy targeting the central nervous system (CNS). However, current delivery methods are either invasive or cause significant systemic exposure. Intranas...Adeno-associated viruses (AAVs) hold significant promise for gene therapy targeting the central nervous system (CNS). However, current delivery methods are either invasive or cause significant systemic exposure. Intranasal (IN) delivery presents a promising noninvasive alternative for direct CNS targeting, though its efficacy in delivering AAVs to the brain has seldom been explored. Here, we quantitatively assessed AAV transduction in the brain and peripheral organs of Swiss, BALB/c, and C57BL/6 J mice following IN administration, using intravenous (IV) injection as a benchmark for comparison. Our findings revealed that IN administration of the AAV9 vector achieved approximately 15% of the transduction efficiency and 9% of the gene expression levels observed with IV delivery. Importantly, IN delivery significantly reduced systemic exposure to most major peripheral organs by up to 1.34 × 10-fold compared to IV injection. The ratios of gene transduction between the brain and various peripheral tissues were calculated, revealing that for key organs such as the liver, stomach, kidney, and spleen, IN delivery achieved higher brain-to-peripheral transduction ratios than IV delivery. These findings underscore the potential of IN delivery for noninvasive brain-targeted gene delivery with significant reductions in peripheral exposure.
Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mito...Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.
Duchenne Muscular Dystrophy is a rare, X-linked neuromuscular disorder that leads to progressive muscle degeneration, loss of ambulation, and premature mortality due to respiratory and cardiac failure. Historically, Duch...Duchenne Muscular Dystrophy is a rare, X-linked neuromuscular disorder that leads to progressive muscle degeneration, loss of ambulation, and premature mortality due to respiratory and cardiac failure. Historically, Duchennke Muscular Dystrophy has been managed through supportive and symptomatic treatments, with limited options for disease modification. However, advancements in gene therapy have introduced promising interventions aimed at addressing the underlying dystrophin deficiency. Delandistrogene moxeparvovec (Elevidys) received accelerated approval from the U.S. Food and Drug Administration in June 2023 for ambulatory children aged 4-5 years with a confirmed diagnosis of Duchenne Muscular Dystrophy. This approval represented an advancement, offering a disease-modifying therapy at an early stage when muscle function remains relatively preserved. The Food and Drug Administration expanded its approval in June 2024 to include both ambulatory and non-ambulatory children aged 4 years and older. This study provides a retrospective real-world analysis of eight Duchenne Muscular Dystrophy patients who received Elevidys gene therapy at our center in Qatar. Recognizing the complexities involved in treating older Duchenne Muscular Dystrophy patients, a standardized protocol for pre- and post-infusion care was implemented. Our findings highlight the positive clinical outcomes of gene therapy for Duchenne Muscular Dystrophy patients in Qatar.
Antibody-drug conjugates (ADCs) are a promising class of targeted cancer therapeutics, but their efficacy is often limited by off-target toxicity caused by payload leakage after internalization into tumor cells. To overc...Antibody-drug conjugates (ADCs) are a promising class of targeted cancer therapeutics, but their efficacy is often limited by off-target toxicity caused by payload leakage after internalization into tumor cells. To overcome this, we developed an ADC alternative using an antibody-guided adeno-associated virus (AAV) vector system that delivers suicide genes specifically to cancer cells. By displaying Protein A on the AAV VP2 capsid, we enabled IgG binding and stable complex formation on the capsid, leading to efficient antibody-guided transduction under our assay conditions. This modular platform allows flexible retargeting specific tumor-associated antigens simply by changing the antibody, without genetic re-engineering of the capsid. Using an AAV2 heparan sulfate binding knockout (HBKO) background to minimize nonspecific infection, we achieved antigen-specific transduction for multiple targets including CD20, EGFR, PSMA, CEA, and CD5 (with varying levels of enhancement depending on the target). In vitro, the system successfully directed EGFP expression and, upon delivery of the pro-apoptotic gene BAX, induced selective apoptosis in target-positive cells. Unlike conventional ADCs, this strategy is designed to minimize the risk of extracellular payload leakage and to confine cytotoxicity primarily to transduced cells, thereby supporting more selective tumor targeting. Our approach may offer a versatile alternative for targeted cancer therapy, with the potential for further development into customizable and precision-guided gene-based treatments.
Commercial development of gene therapeutics often requires transitioning to human payload genes as initial proof-of-concept studies in animal models often use taxa-specific orthologs. Such transitions also provide opport...Commercial development of gene therapeutics often requires transitioning to human payload genes as initial proof-of-concept studies in animal models often use taxa-specific orthologs. Such transitions also provide opportunities to address potential secondary structure and immune-related subsequences as with human Smad7 cDNA, which was optimized by removing several repeats, potential hairpins and negative cis elements. Thermodynamic modeling at or above minimal free energy states revealed substantial improvements in secondary structure with fewer hairpins and improved diversity scores. Serotype 6 adeno-associated viral vectors with optimized human Smad7 (AVGN7.2) expression constructs were equally or more effective than those with wild-type mouse Smad7 in stimulating skeletal muscle hypertrophy and enhancing isometric torque of hind-limb dorsiflexor muscles in vivo. In murine models of Duchenne Muscular dystrophy, where deficits in muscle mass and disproportionate declines in force are pathognomonic, AVGN7.2 proportionally increased muscle mass and isometric torque while normalizing contractile kinetics. Such improvements occurred without deleterious impacts on serum creatine kinase, fibrosis or myofiber central nucleation. These data suggest that AVGN7.2 is capable of enhancing dystrophic muscle function without exacerbating muscle degeneration. Although these functional effects were partial, they resembled those of several dystrophin-targeting drugs and suggest that combinatorial approaches may safely yield further benefit.
Angelman syndrome (AS) is a severe neurodevelopmental disorder most often caused by deletion of the maternally inherited UBE3A allele (matUBE3A). In neurons, a long non-coding antisense RNA (Ube3a-ATS) silences the pater...Angelman syndrome (AS) is a severe neurodevelopmental disorder most often caused by deletion of the maternally inherited UBE3A allele (matUBE3A). In neurons, a long non-coding antisense RNA (Ube3a-ATS) silences the paternally-inherited UBE3A allele (patUBE3A). Here, we find that delivery of a zinc finger nuclease (ZFN) pair targeted to 86 Snord115 genes within Ube3a-ATS (ZFN17/18) using adeno-associated virus (AAV) can unsilence patUBE3A in primary neuron cultures and in the brain of a mouse model of AS for at least 9 weeks. The AAV vector genome integrated at ZFN17/18 on-target sites in cultured neurons and, as evidence of specificity, did not integrate at predicted off-target sites. AAV vectors carrying nickase and catalytically inactive ZFN17/18 variants failed to appreciably unsilence patUbe3a and did not integrate at on-target sites. In vivo, we observed significant knockdown of Ube3a-ATS in AS-model mice, resulting in some neurons reaching UBE3A levels like those of wild-type mice. ZFN17/18 did not downregulate Snrpn, Snord116, or IPW in vivo, genes that are associated with Prader-Willi syndrome. Overall, our findings demonstrate the potential use of multi-target ZFNs as therapeutics for AS.
Sporadic inclusion body myositis (IBM) is a highly debilitating muscle degenerative and rare disease of the middle aged and elderly. Because immunosuppressants fail to prevent muscle wasting in IBM patients and can even...Sporadic inclusion body myositis (IBM) is a highly debilitating muscle degenerative and rare disease of the middle aged and elderly. Because immunosuppressants fail to prevent muscle wasting in IBM patients and can even exacerbate it, drugs like AVGN7.2 are being developed to halt degeneration and to enhance muscle mass and function. AVGN7.2 is a novel gene therapeutic that attenuates activin receptors through muscle-specific human (h) SMAD7 expression and as part of its preclinical development, we performed a 91-day single-dose toxicology assessment of systemic safety, biodistribution and immunogenicity in accordance with Good Laboratory Practices. Standard physiological, ophthalmoscopic, hematological and serum chemistry examinations were performed and no adverse drug-related effects were detected at any dose (2.3E + 13, 7E + 13 and 2.1E + 14 vg/kg), resulting in a No Observed Adverse Effect Level of 2.1e14 vg/kg. Mice mounted early IgM and late IgG responses to the AAV6 capsid, but no response to the hSMAD7 protein. Vector biodistribution mirrored previously published patterns with liver followed by striated muscle having the highest levels, although overexpression of hSMAD7 and the S6RP biomarker only occurred in muscle. These data suggest that AVGN7.2 was well-tolerated even at doses known to elicit clinical toxicities with muscle-tropic AAV capsids other than AAV6.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, with limited treatments. Gene therapy offers an alternative strategy for treating a large portion of ALS patients, however, the disparate genetic...Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, with limited treatments. Gene therapy offers an alternative strategy for treating a large portion of ALS patients, however, the disparate genetic alterations in ALS complicate the development of gene therapies. Tyrosine receptor kinase B (TRKB) and Tyro3 receptors are highly expressed in mouse spinal cord motor neurons, suggesting that their ligands, brain-derived neurotrophic factor (BDNF) and growth arrest-specific 6 (GAS6), respectively, are crucial for neuronal survival. In this study, we tested whether genetically induced and muscle tissue-specific expression of such survival-enhancing ligands would ameliorate symptom development in the SOD1 ALS mouse model. The therapeutic vectors (AAV-P7-HuBDNF-teLuc or AAV-P7-HuGAS6), or a control vector (AAV-P7-teLuc) were injected intravenously via the retro-orbital route and intramuscularly into the hindlimb skeletal muscle of six-week-old mice. Treatment with the therapeutic vectors delayed disease onset and slowed progression in both male and female mice. Interestingly, a sex-specific response was observed, with female mice benefiting more from the treatments than males. Lumbar motor neuron survival was more sustained in the therapeutic vector-treated group compared to control vector group. No statistically significant extension of lifespan was observed in the treated groups.
The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorde...The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.
Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mo...Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mortality. While established lipid-lowering therapies, primarily statins, are effective for many patients, a significant proportion either fail to achieve optimal LDL-C targets, experience dose-limiting side effects, or face challenges with the long-term adherence required for sustained cardiovascular benefit. The recent emergence and rapid advancement of precise gene editing technologies most notably CRISPR-Cas9 and its advanced variants like base editing and prime editing offer a revolutionary therapeutic paradigm. These tools have the potential to achieve durable modification of the expression or function of genes fundamentally involved in cholesterol metabolism. This comprehensive overview integrates the current knowledge of critical cholesterol regulatory pathways and the main protein targets that are suitable for gene editing. The fundamental mechanisms, relative advantages, and inherent limitations of gene editing platforms and delivery systems for clinical translation are examined. The expanding preclinical data and groundbreaking clinical evidence highlighting the transformative potential of gene editing to achieve significant and lasting reductions in LDL-C, especially through promising therapies like VERVE base editors targeting PCSK9 and ANGPTL3 are critically evaluated. The challenges including off-target effects, delivery efficiency and specificity, long-term safety and durability, complex ethical considerations, and evolving regulatory landscapes that must be rigorously navigated for these therapies to become mainstream clinical practice are thoroughly addressed. Successfully overcoming these challenges could mark the beginning of a new era of personalized, one-time treatments for hypercholesterolemia.
Guidance from the U.S. Food and Drug Administration and other regulatory agencies recommends long-term follow-up (LTFU) studies of gene therapy recipients. The primary objective of LTFU studies is to understand the long-...Guidance from the U.S. Food and Drug Administration and other regulatory agencies recommends long-term follow-up (LTFU) studies of gene therapy recipients. The primary objective of LTFU studies is to understand the long-term safety of gene therapy products; evaluation of efficacy outcomes may be a secondary goal. To learn more about LTFU study design and conduct, we conducted a descriptive study of key characteristics of LTFU gene therapy study protocols registered in ClinicalTrials.gov, including data about status and duration, funding source, enrollment, number of clinical trial sites per study, eligibility criteria, geographic location, and intent to monitor and report adverse events. This analysis enabled a better understanding of how registered LTFU studies are currently designed and stimulated ideas for improvement, which are discussed. Most notably, our results suggest that there is a lack of harmonization in how safety outcomes are monitored and reported across LTFU studies. Standardization and/or harmonization of outcome reporting for LTFU studies of GTs may increase their scientific value. The development of better guidance and innovative approaches for LTFU study design and conduct would help support best practices and the fulfillment of LTFU commitments to understand the overall long-term benefit-risk profile of GT products.