Clinical Potential Of Gene Therapy Research Articles

Lentiviral Mediated Gene Therapy for Pyruvate Kinase Deficiency: A Global Phase 1 Study for Adult and Pediatric Patients

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18-<50 years), followed by 2 older pediatric subjects (age ≥12-17 years) once initial safety has been established. Subsequently, 2 younger pediatric subjects (age ≥8-11 years) will be enrolled. Peripheral blood (PB) hematopoietic stem cells are collected on two consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following final release testing of the investigational product (IP), RP-L301, myeloablative conditioning with therapeutic drug monitoring (TDM)-guided busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis. Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Self-Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery.

An ideal vector in gene therapy should exhibit high serum stability, excellent biocompatibility, a desired transfection efficacy and permeability into targeted tissues. Here, we describe a class of low-molecular-weight fluorodendrimers for efficient gene delivery. These materials self-assemble into uniform nanospheres and allow for efficient transfection at low charge ratios and very low DNA doses with minimal cytotoxicity. Our results demonstrate that these vectors combine the features of synthetic gene vectors such as liposomes and cationic polymers and present promising potential for clinical gene therapy.

Self‐Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery

AbstractAn ideal vector in gene therapy should exhibit high serum stability, excellent biocompatibility, a desired transfection efficacy and permeability into targeted tissues. Here, we describe a class of low‐molecular‐weight fluorodendrimers for efficient gene delivery. These materials self‐assemble into uniform nanospheres and allow for efficient transfection at low charge ratios and very low DNA doses with minimal cytotoxicity. Our results demonstrate that these vectors combine the features of synthetic gene vectors such as liposomes and cationic polymers and present promising potential for clinical gene therapy.

Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors.

Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.

Clinical potential of gene therapy: towards meeting the demand

Since the discovery that new genetic material could be transferred into human cells resulting in induced expression of genes and proteins, clinicians and scientists have been working to harness the technology for clinical outcomes. This article provides a summary of the current status of developments within the broad discipline of clinical gene therapy. In pursuing the treatment of diverse clinical conditions, a wide variety of therapeutics, each tailor-made, may be required. Gene therapy offers the possibility of accurately and specifically targeting particular genetic abnormalities through gene correction, addition or replacement. It represents a compelling idea that adds a new dimension to our portfolio of credible therapeutic choices.

Peptide-Based Vector of VEGF Plasmid for Efficient Gene Delivery in Vitro and Vessel Formation in Vivo

Critical limb ischemia is regarded as a potentially lethal disease, and the treatment effects of existing therapies are limited. Here, in order to develop a potential approach to improve the therapy effects, we designed a peptide of TAT-PKKKRKV as the vector for VEGF165 plasmid to facilitate in vivo angiogenesis. In in vitro studies, TAT-PKKKRKV with low cytotoxicity exhibited efficient transfection ability either with or without serum. Additionally, application of TAT-PKKKRKV/VEGF165 complexes in hindlimb ischemia rats obviously promoted the expression of VEGF protein, which further enhanced effective angiogenesis. The results indicated that TAT-PKKKRKV is an efficient gene vector with low toxicity both in vitro and in vivo, which has great potential for clinical gene therapy.

Pulmonary delivery of nucleic acids

The lung is an appropriate present and future target for gene therapy approaches designed to treat inherited monogenic diseases, eradicate bronchial tumours, transfer pharmacologically active products to the general circulation, express enzymes to catabolise toxins, manage pulmonary hypertension and lung injury and vaccinate against infection. Despite 35 years of gene therapy research and some significant milestones in molecular biology, the clinical potential of gene therapy has yet to be realised. In pulmonary gene therapy the nucleic acid cargo needs to be delivered to cells in the �target region of the lung, and even in cases when these targets are well defined this is severely limited by the pulmonary architecture, clearance mechanisms, immune activation, the presence of respiratory mucus and the availability of a truly representative biological model. The challenge from a drug delivery perspective is to consider the suitability of conventional nebulisers and inhalers for delivering DNA to the lung and design and apply integrated formulation and device solutions specific to nucleic acid delivery.

705. AMD3100-Mobilized CD34+ Cells Are Phenotypically Different and Better Targets for Retroviral Transduction Than G-CSF-Mobilized CD34+ Cells in Rhesus Macaques

Successful ex vivo and in vivo expansion of transgene modified hematopoietic stem cells (HSC) have great application potential for clinical gene therapy. Overexpression of the human HoxB4 gene induces ex vivo expansion and self-renewal of murine HSC. Since mouse and human differ substantially, we have started to study the effect of HoxB4 on HSC in a large animal model. Here we investigated if human HoxB4 enhances ex vivo expansion and in vivo selection of CD34+ cells from mobilized dog peripheral blood (PB), cytokine primed baboon and macaque bone marrow (BM), human cord blood (CB) and mobilized human PB. CD34+ cells were transduced with Phoenix-GALV or RD114-pseudotype MSCV-HoxB4-ires-GFP or the control vector, MSCV-ires-YFP. After 4 weeks culture, the percentage of HoxB4-overexpressing cells significantly increased for dog, baboon and macaque cells. HoxB4 also imparts a noteworthy effect on expansion of progenitors. HoxB4 expression resulted in an up to 2-fold expansion for human PB, 4-fold expansion for human CB, 5 to 7-fold expansion for baboon and macaque, and 28-fold expansion for dog cells in CFUs when compared with YFP-transduced cells. Furthermore, in vivo competitive repopulation experiments in NOD/SCID mouse model were performed to test the effects of HoxB4 on HSC. The engraftment level for GFP+ cells was 6-fold higher for PB cells, and 10-fold higher for CB cells compared with YFP+ cells, indicating a significant growth advantage of HoxB4-transduced cells. To examine whether HoxB4 expression promotes expansion of HSC in large animal model, myeloblated dogs were transplanted with HoxB4- and YFP- transduced cells. Preliminary data show that the ratio of GFP+ cells to YFP+ cells increased from 0.9 at day 0 to ratio of 9 at day 20 post-transplant, demonstrating a remarkable in vivo selection of HoxB4-transduced cells in clinically relevant model. To explore the mechanisms that underlie the apparently more pronounced effects on cells from dog, macaque and baboon compared to human cells, we monitored the HoxB4 expression in these cells at the RNA level by real-time RT-PCR and at the protein level by western blot. Comparable level of HoxB4 and YFP transcripts was detected in dog and mouse transduced cells, while copies of HoxB4 transcripts were only 10–30% those of YFP transcripts for human and baboon transduced cells. Likewise, HoxB4 protein expression in human and baboon cells decreased continuously with time, in parallel with the differentiation of these cells. However, protein expression in transduced mouse and dog cells was stable, which was associated with inhibition of cell differentiation. We also found that HoxB4 was tyrosine phosphorylated in transduced mouse and dog cells. Our results suggest that the higher level of HoxB4 protein expressed inhibit the differentiation and promote self-renewal of HSC, while lower level confers a less pronounced effects on self-renewal. Further insights into the regulation of transduced HoxB4 in human cells might pave the road to its successful application in clinical gene therapy.

Recent advances in the pharmacology of neurological gene therapy.

The choice of vectors, transgenes, regulatory elements, delivery approaches and the capacity to transduce the appropriate target cell type all influence the effectiveness of gene therapy for neurological diseases. Furthermore, even if many strategies are sound and effective in experimental animals, issues relating to side effects of gene therapy, longevity of therapeutic transgene expression and diffusion throughout the brain can limit the clinical potential of gene therapy. During the past 12-18 months, there have been significant advances in the following areas: the capacity to target vectors to predetermined cells types; the development of gene therapy approaches for the treatment of dominant inherited and neurodegenerative diseases; the capacity to achieve systemic delivery of viral vectors to the brain; and the development of viral vectors to model neurological diseases.

Theory and in vivo application of electroporative gene delivery.

Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

Gene therapy using retroviral vectors.

Gene therapy is a novel approach for treating various congenital and acquired genetic disorders, including cancer, heart disease, and acquired immune deficiency syndrome. Amongst possible gene delivery systems, retroviral vector mediated gene transfer has been the most extensively studied and has been approved for use in over 40 current Phase I/II clinical trials for the treatment of various disorders, primarily cancers. Recent technological improvements include the optimization of vector production by concentration and lyophilization, resulting in high titers of vectors, as well as the large-scale production of vector-produced cells for the treatment of brain cancer. Present clinical protocols require specialized care centers with expertise in molecular biology and cell transplantation. Considerable effort is under way to develop retroviral vectors that can be both injected directly into the body and targeted to specific cell types within the body. Such vectors could be administered to patients by physicians in their offices. Successful development of this new technology would greatly expand the clinical potential of gene therapy.

Development in Somatic Gene Therapy

Clinical trials of gene transfer have begun to validate the clinical potential of gene therapy. With this progress, attention has turned increasingly to analysing the ability of various methods of gene therapy to satisfy clinical and commercial needs. There are fundamental differences between cell-based therapies, which employ genetically modified cells as a therapeutic product, virus-based therapies, which employ recombinant viral vectors as a therapeutic product and gene-based therapies in which DNA itself is formulated as a pharmaceutical product, in terms of the technologies required to develop and produce the product, the mode of clinical application, the clinical risks and the economics of commercialisation. This report reviews the potential indications for gene therapy and the status of various approaches to gene therapy in development in the context of a field which is moving rapidly from basic research to product development.