Current and emerging gene therapies for haemophilia A and B.

INTRODUCTION: After decades of stumbling clinical development, the first gene therapies for haemophilia A and B have been commercialized and have normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several other clinical programs testing adeno-associated viral (AAV) vector gene therapy are at various stages of clinical testing. DISCUSSION: Multiyear follow-up in phase 1/2 and 3 studies showed long-term and sometimes curative but widely variable and unpredictable efficacy. Liver toxicities, mostly low-grade, occur in the 1st year in at least some individuals in all haemophilia A and B trials and are poorly understood. Wide variability and unpredictability of outcome and slow decline of FVIII levels are a major disadvantage because immune responses to AAV vectors preclude repeat dosing, which otherwise could improve suboptimal or restore declining expression, while overexpression may predispose to thrombosis. Long-term safety outcomes will need lifelong monitoring because AAV vectors infused at high doses integrate into chromosomes at rates that raise questions about potential oncogenicity and necessitate vigilance. Alternative gene transfer systems employing gene editing and/or non-viral vectors are under development and promise to overcome some limitations of the current state of the art for both haemophilia A and B. CONCLUSIONS: AAV gene therapies for haemophilia have now become new treatment options but not universal cures. AAV is a powerful but imperfect gene transfer platform. Biobetter FVIII transgenes may help solve some problems plaguing gene therapy for haemophilia A. Addressing variability and unpredictability of efficacy, and delivery of gene therapy to ineligible patient subgroups may require different gene transfer systems, most of which are not ready for clinical translation yet but bring innovations needed to overcome the current limitations of gene therapy.

Promoter Optimization Circumvents Bcl-2 Transgene-Mediated Suppression of Lentiviral Vector Production.

Lentiviral vectors are a robust gene delivery tool for inducing transgene expression in a variety of cells. They are well suited to facilitate the testing of therapeutic candidate genes in vitro, due to relative ease of packaging and ability to transduce dividing and non-dividing cells. Our goal was to identify a gene that could be delivered to the heart to protect against cancer-therapy-induced cardiotoxicity. We sought to generate a lentivirus construct with a ubiquitous CMV promoter driving expression of B-cell lymphocyte/leukemia 2 gene (Bcl-2), a potent anti-apoptotic gene. Contrary to our aim, overexpression of Bcl-2 induced cell death in the producer HEK293T cells, resulting in failure to produce usable vector titre. This was circumvented by exchanging the CMV promoter to the cardiac-specific NCX1 promoter, leading to the successful production of a lentiviral vector which could induce cardioprotective expression of Bcl-2. In conclusion, reduced expression of Bcl-2 driven by a weaker promoter improved vector yield, and led to the production of functional cardioprotective Bcl-2 in primary cardiomyocytes.

Gene therapy in cancer.

Gene therapy, recently frequently investigated, is an alternative treatment method that introduces therapeutic genes into a cancer cell or tissue to cause cell death or slow down the growth of the cancer. This treatment has various strategies such as therapeutic gene activation or silencing of unwanted or defective genes; therefore a wide variety of genes and viral or nonviral vectors are being used in studies. Gene therapy strategies in cancer can be classified as inhibition of oncogene activation, activation of tumor suppressor gene, immunotherapy, suicide gene therapy and antiangiogenic gene therapy. In this review, we explain gene therapy, gene therapy strategies in cancer, approved gene medicines for cancer treatment and future of gene therapy in cancer. Today gene therapy has not yet reached the level of replacing conventional therapies. However, with a better understanding of the mechanism of cancer to determine the right treatment and target, in the future gene therapy, used as monotherapy or in combination with another existing treatment options, is likely to be used as a new medical procedure that will make cancer a controllable disease.

A systematic review of adeno-associated virus gene therapy clinical trials for HIV - A potential solution for patients with haemophilia and HIV?

INTRODUCTION: In the past HIV infection was a common complication of haemophilia therapy. Gene therapy trials in Haemophilia patients using rAAV have shown promising results; Unfortunately, the majority of gene therapy trials studies have excluded HIV positive patients. We decided to systematically review the published clinical trials using rAAV for HIV prevention. METHODS: A comprehensive literature search was performed to identify studies evaluating clinical trials using rAAV for HIV. The search was conducted using the MEDLINE/PubMed databases. Search keywords included 'gene therapy', 'adeno-associated virus', 'HIV' and 'clinical trial'. RESULTS: Three studies met our inclusion criteria. Two were phase 1 studies and one was a phase 2 study. One study examined an AAV coding for human monoclonal IgG1 antibody whereas the other two studies delivered a vector coding for viral protease and part of reverse transcriptase. All studies administered the vaccine intramuscularly and showed a response as well a good safety profile. DISCUSSION: The concept of using a viral vector to prevent a viral infection is revolutionary. Due to the paucity of information regarding application of any gene therapy in HIV patients and the potential use of gene therapy in haemophilia patients with HIV in the future warrants attention.

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.).

Gene therapy for hemophilia.

The cloning of the factor VIII (FVIII) and factor IX (FIX) genes in the 1980s has led to a succession of clinical advances starting with the advent of molecular diagnostic for hemophilia, followed by the development of recombinant clotting factor replacement therapy. Now gene therapy beckons on the back of decades of research that has brought us to the final stages of the approval of 2 products in Europe and United States, thus heralding a new era in the treatment of the hemophilias. Valoctocogene roxaparvovec, the first gene therapy for treatment of hemophilia A, has been granted conditional marketing authorization in Europe. Another approach (etranacogene dezaparvovec, AMT-061) for hemophilia B is also under review by regulators. There are several other gene therapy approaches in earlier stages of development. These approaches entail a one-off infusion of a genetically modified adeno-associated virus (AAV) engineered to deliver either the FVIII or FIX gene to the liver, leading to the continuous endogenous synthesis and secretion of the missing coagulation factor into the circulation by the hepatocytes, thus preventing or reducing bleeding episodes. Ongoing observations show sustained clinical benefit of gene therapy for >5 years following a single administration of an AAV vector without long-lasting or late toxicities. An asymptomatic, self-limiting, immune-mediated rise in alanine aminotransferase is commonly observed within the first 12 months after gene transfer that has the potential to eliminate the transduced hepatocytes in the absence of treatment with immunosuppressive agents such as corticosteroids. The current state of this exciting and rapidly evolving field, as well as the challenges that need to be overcome for the widespread adaptation of this new treatment paradigm, is the subject of this review.

Functional Intercellular Transmission of miHTT via Extracellular Vesicles: An In Vitro Proof-of-Mechanism Study.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by GAG expansion in exon 1 of the huntingtin (HTT) gene. AAV5-miHTT is an adeno-associated virus serotype 5-based vector expressing an engineered HTT-targeting microRNA (miHTT). Preclinical studies demonstrate the brain-wide spread of AAV5-miHTT following a single intrastriatal injection, which is partly mediated by neuronal transport. miHTT has been previously associated with extracellular vesicles (EVs), but whether EVs mediate the intercellular transmission of miHTT remains unknown. A contactless culture system was used to evaluate the transport of miHTT, either from a donor cell line overexpressing miHTT or AAV5-miHTT transduced neurons. Transfer of miHTT to recipient (HEK-293T, HeLa, and HD patient-derived neurons) cells was observed, which significantly reduced HTT mRNA levels. miHTT was present in EV-enriched fractions isolated from culture media. Immunocytochemical and in situ hybridization experiments showed that the signal for miHTT and EV markers co-localized, confirming the transport of miHTT within EVs. In summary, we provide evidence that an engineered miRNA-miHTT-is loaded into EVs, transported across extracellular space, and taken up by neighboring cells, and importantly, that miHTT is active in recipient cells downregulating HTT expression. This represents an additional mechanism contributing to the widespread biodistribution of AAV5-miHTT.

Immune Responses and Immunosuppressive Strategies for Adeno-Associated Virus-Based Gene Therapy for Treatment of Central Nervous System Disorders: Current Knowledge and Approaches.

Adeno-associated viruses (AAVs) are being increasingly used as gene therapy vectors in clinical studies especially targeting central nervous system (CNS) disorders. Correspondingly, host immune responses to the AAV capsid or the transgene-encoded protein have been observed in various clinical and preclinical studies. Such immune responses may adversely impact patients' health, prevent viral transduction, prevent repeated dosing strategies, eliminate transduced cells, and pose a significant barrier to the potential effectiveness of AAV gene therapy. Consequently, multiple immunomodulatory strategies have been used in attempts to limit immune-mediated responses to the vector, enable readministration of AAV gene therapy, prevent end-organ toxicity, and increase the duration of transgene-encoded protein expression. Herein we review the innate and adaptive immune responses that may occur during CNS-targeted AAV gene therapy as well as host- and treatment-specific factors that could impact the immune response. We also summarize the available preclinical and clinical data on immune responses specifically to CNS-targeted AAV gene therapy and discuss potential strategies for incorporating prophylactic immunosuppression regimens to circumvent adverse immune responses.

Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Gene therapy - are we ready now?

INTRODUCTION: Haemophilia therapy has evolved from rudimentary transfusion-based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno-associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals. DISCUSSION: Phase 3 studies along with multiyear follow-up in earlier phase investigations raised questions about efficacy as well as short- and long-term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low-grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long-term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance. CONCLUSIONS: AAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Gene therapy: Comprehensive overview and therapeutic applications.

Gene therapy is the product of man's quest to eliminate diseases. Gene therapy has three facets namely, gene silencing using siRNA, shRNA and miRNA, gene replacement where the desired gene in the form of plasmids and viral vectors, are directly administered and finally gene editing based therapy where mutations are modified using specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short tandem repeats (CRISPR)/CRISPR-associated protein (Cas)-associated nucleases. Transfer of gene is either through transformation where under specific conditions the gene is directly taken up by the bacterial cells, transduction where a bacteriophage is used to transfer the genetic material and lastly transfection that involves forceful delivery of gene using either viral or non-viral vectors. The non-viral transfection methods are subdivided into physical, chemical and biological. The physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound and hydrodynamic gene transfer. The chemical methods utilize calcium- phosphate, DAE-dextran, liposomes and nanoparticles for transfection. The biological methods are increasingly using viruses for gene transfer, these viruses could either integrate within the genome of the host cell conferring a stable gene expression, whereas few other non-integrating viruses are episomal and their expression is diluted proportional to the cell division. So far, gene therapy has been wielded in a plethora of diseases. However, coherent and innocuous delivery of genes is among the major hurdles in the use of this promising therapy. Hence this review aims to highlight the current options available for gene transfer along with the advantages and limitations of every method.

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.

Gene Therapy for Critical Limb Ischemia: Per Aspera ad Astra.

Peripheral artery diseases remain a serious public health problem. Although there are many traditional methods for their treatment using conservative therapeutic techniques and surgery, gene therapy is an alternative and potentially more effective treatment option especially for "no-option" patients. This review treats the results of many years of research and application of gene therapy as an example of treatment of patients with critical limb ischemia. Data on successful and unsuccessful attempts to use this technology for treating this disease are presented. Trends in changing the paradigm of approaches to therapeutic angiogenesis are noted: from viral vectors to non-viral vectors, from gene transfer to the whole organism to targeted transfer to cells and tissues, from single-gene use to combination of genes; from DNA therapy to RNA therapy, from in vivo therapy to ex vivo therapy.

Hemophilia gene therapy: ushering in a new treatment paradigm?

After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.

Targeting Human Osteoarthritic Chondrocytes with Ligand Directed Bacteriophage-Based Particles.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs.