Safety Of Gene Therapy Research Articles

MicroRNA-regulated viral vectors for gene therapy.

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

Improving Lentiviral Transduction Efficiency with Microfluidic Systems

Background: Recent clinical trials have demonstrated the efficacy and safety of gene therapy utilizing HIV-derived lentiviral vectors (LVs) for blood disorders. However, the LV requirements and clinical ex vivo cell transduction protocols used in these studies exposes the limitations of the technology and beckons the need for improved LV manufacturing and clinical transduction efficiency. Many methods have been devised to enhance efficiency, although none have circumvented the exorbitant amounts of virus required to achieve therapeutic HSC transduction. Furthermore, prolonged ex vivo cell culture is necessary to achieve sufficient transduction despite exposure to toxic byproducts of LV production. To that end, we developed a novel, scalable microfluidic for clinical LV transduction that leverages mass transfer principles to significantly reduce the amount of LV required to achieve therapeutic levels of gene transfer and transduction time by more efficiently exposing cells to virus.Results: Jurkats were transduced with a GFP-encoding clinical LV in microfluidics with surface areas (SAs) comparable to the bottom surface of 96 and 6-well plates. Microfluidic transductions were compared to well plate transductions with matched SA, cell numbers, viral particles, and incubation times. After LV incubation, cells were removed from the microfluidics and well plates, spun down, re-suspended with fresh media, and cultured for at least 72 hours at 37°C and 5% CO2. Cells were assessed for GFP expression with flow cytometry. Preliminary mouse studies utilized Sca+ cells isolated from CD45.1 donor mice via positive selection. The cells were transduced in the scaled up microfluidic with a bioengineered coagulation factor VIII (fVIII) transgene encoding LV and transplanted into host hemophilia A mice after myeloablative conditioning. Two weeks post-transplantation, blood samples were taken from the recipient animals and assayed for donor cell engraftment by flow cytometry and plasma fVIII activity by chromogenic assay.The high SA:volume ratio of the microfluidic enhances transduction by physically bringing cells and virus into closer proximity and enabling high concentrations of virus to be used without increasing the amount of virus set by the minimum volume requirements of LV transduction platforms (Fig. 1A). The polystyrene bottom of the microfluidic allows for Retronectin coating that immobilizes non-adherent cells on the bottom surface. LV can then be perfused at low concentrations to maintain a constant supply of fresh virus to the cells, increase convective mixing, and to minimize cell exposure to the toxic byproducts of LV production (Fig. 1B). These microfluidics have been scaled up to accommodate 106 cells, with potential to scale up to 107-108 cells (Fig. 1C). Cells transduced in the microfluidics showed 2-6 fold increases in GFP expression over well plates utilizing the same amount of cells, virus, and incubation times (Fig. 2A). The kinetics of LV transduction in the microfluidics also are faster, as seen by the steeper transduction curve. Five hours of incubation in the microfluidic yielded comparable transduction to 24 hours in the 6-well plate (Fig. 2B). Improvements in transduction also were observed by perfusing virus despite using lower virus concentrations (Fig. 2C). Finally, hemophilia A mice transplanted with donor CD45.1 Sca+ cells transduced in the microfluidic have engrafted (Fig. 3A) and produce fVIII (Fig. 3B) after two weeks with similar profiles to control cells transduced in a 6-well plate despite using half the amount of virus and shorter incubation times.Conclusions and ongoing efforts: We describe a novel microfluidic that significantly reduces the amount of virus and ex vivo processing time required for therapeutic levels of transduction in clinical gene therapy. This device is versatile in its compatibility with current transduction strategies such as Retronectin and polybrene in addition to offering new approaches to boosting gene transfer efficiency. Furthermore, we have shown that the device has clinical potential by successfully scaling up cell numbers and transplanting mice with microfluidic transduced cells, of which there is an ongoing effort to monitor fVIII production and determine virus copy number. Future work will involve optimization with transduction-enhancing compounds, further scaling, and continued in vivo experiments. [Display omitted] [Display omitted] [Display omitted] DisclosuresSpencer:Expression Therapeutics: Equity Ownership. Doering:Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership.

Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. DisclosuresNo relevant conflicts of interest to declare.

A Graph Based Framework to Model Virus Integration Sites

With next generation sequencing thousands of virus and viral vector integration genome targets are now under investigation to uncover specific integration preferences and to define clusters of integration, termed common integration sites (CIS), that may allow to assess gene therapy safety or to detect disease related genomic features such as oncogenes.Here, we addressed the challenge to: 1) define the notion of CIS on graph models, 2) demonstrate that the structure of CIS enters in the category of scale-free networks and 3) show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD) as a testing dataset.

Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

Progresses towards safe and efficient gene therapy vectors.

The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.

Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs.

Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery.

The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

Abstract 292: Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma

Abstract Electrochemotherapy is now well established ablative local tumor treatment for veterinary as well as human cancer. Electrochemotherapy combines the use of permeabilizing electric pulses applied directly at the tumor site with the injection of chemotherapeutic drugs, cisplatin or bleomycin. Increased membrane permeability enables enhanced entry of chemotherapeutic drug into the cells and resulted in higher tumor cell kill. Up to 80% complete responses are obtained after single electrochemotherapy treatment of cutaneous tumors of different histology (Yarmush et al. Annu Rev Biomed Eng. 2014; 16:295-320). To add a systemic component to this very effective local treatment, in preclinical studies it was combined with several different immune therapies, including gene therapy with plasmid DNA encoding interleukin-12 (IL-12) (Radiol Oncol. 2012;46:302-11). In our on-going clinical research, the effect of IL-12 gene therapy combined with electrochemotherapy was evaluated in 18 client- owned dogs with mastocytomas. Written consent for participation was obtained from the owners and the study was approved by the competent authorities (Veterinary administration and Administration for biotechnology of the Ministry of agriculture and environment; Republic Ethical Committee for the experiments involving animals). Electrochemotherapy using intratumoral injection of bleomycin and application of electric pulses was preformed prior to gene therapy. Plasmid encoding IL-12 was injected peritumorally into the skin and immediately thereafter electric pulses were applied. Local response of tumors was evaluated by measurements of tumors’ size. Systemic response was assessed by determination of IL-12 and IFN-γ in patients’ sera. Safety of gene therapy was evaluated by qRT-PCR measurement for the presence of plasmid DNA at the site of application and possible horizontal gene transfer by in vitro transformation of plasmid DNA encoding IL-12 into the bacteria isolated from the dog's (patient’s) skin. One month after the therapy, 11 dogs (61%) had a complete response, 4 (22%) partial response, and 3 (16%) stable disease. At the end of observation period (median 25 months) 16 dogs (89%) had complete response and 2 had to be euthanized due to the progression of disease. In all dogs, except two, serum IFN-γ or IL-12 levels were detected, without systemic toxicity. At the site of DNA plasmid application, except for one patient, the plasmid was not detected at 1 week post-treatment. No horizontal gene transfer was detected; plasmid was not detected in any of the residential bacteria and in vitro transformation of plasmid into isolated strains was negative. Collectively, the results of our on-going clinical trial demonstrate that IL-12 gene therapy combined with electrochemotherapy is safe, feasible and effective treatment for canine mastocytoma. Citation Format: Maja Cemazar, Jerneja Ambrozic Avgustin, Gregor Sersa, Darja Pavlin, Ana Krhac Levacic, Natasa Tesic, Mitja Rak, Ursa Lampreht, Natasa Tozon. Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 292. doi:10.1158/1538-7445.AM2015-292

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.

Gene therapy and peripheral nerve repair: a perspective.

Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan’s, Parkinson’s (PD) and Alzheimer’s disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV) and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS) repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS-gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study.

Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy.

Recently, adenovirus (Ad) has been utilized as a viral vector for efficient gene delivery. However, substantial immunogenicity and toxicity have obstructed oncolytic Ad's transition into clinical studies. The goal of this study is to generate an adenoviral vector complexed with multidegradable bioreducible core-cross-linked polyethylenimine (rPEI) polymer that has low immunogenicity and toxicity while having higher transduction efficacy and stability. We have synthesized different molecular weight rPEIs and complexed with Ad at varying molar ratios to optimize delivery of the Ad/polymer complex. The size and surface charge of Ad/rPEIs were characterized. Of note, Ad/rPEIs showed significantly enhanced transduction efficiency compared to either naked Ad or Ad/25 kDa PEI in both coxsackievirus and adenovirus receptor (CAR) positive and negative cancer cells. The cellular uptake result demonstrated that the relatively small size of Ad/16 kDa rPEIs (below 200 nm) was more critical to the complex's internalization than its surface charge. Cancer cell killing effect and viral production were significantly increased when oncolytic Ad (RdB/shMet, or oAd) was complexed with 16 kDa rPEI in comparison to naked oAd-, oAd/25 kDa PEI-, or oAd/32 kDa rPEI-treated cells. This increased anticancer cytotoxicity was more readily apparent in CAR-negative MCF7 cells, implying that it can be used to treat a broad range of cancer cells. Furthermore, A549 and HT1080 cancer cells treated with oAd/16 kDa rPEI had significantly decreased Met and VEGF expression compared to either naked oAd or oAd/25 kDa PEI. Overall, these results demonstrate that shMet expressing oncolytic Ad complexed with multidegradable bioreducible core-cross-linked PEI could be used as efficient and safe cancer gene therapy.

289. Safe and Effective Gene Therapy for Wiskott – Aldrich Syndrome Using an Insulated Lentiviral Vector

Wiskott–Aldrich Syndrome (WAS) is a severe X-linked immunodeficiency disorder characterized by recurrent infection, thrombocytopenia, and autoimmunity resulting from mutations within the WAS gene. The encoded gene product, WAS protein (WASp), is a scaffold protein important for activation of the actin cytoskeleton downstream of multiple hematopoietic receptors. Development of safe and effective hematopoietic stem cell (HSC) gene therapy for WAS would provide a valuable treatment alternative to allogeneic bone marrow transplantation. Of critical importance for such an approach is the capacity to achieve sufficient WASp expression in relevant lineages to correct the multiple deficits, while avoiding development of autoimmune disease. We previously tested a panel of self-inactivating (SIN) lentiviral (LV) vectors expressing human WAS via alternative internal promoters and observed optimal expression and rescue using the modified γ-retroviral derived LTR, MND. To improve the potential safety and stability of expression, we incorporated a 650bp chimeric insulator from the chicken β-globin locus into this LV. Using in vitro assays the insulated LV exhibited minimal LMO2 transactivation and no evidence for immortalization of cultured HSC. Next, we generated a stable high-titer producer clone expressing this candidate clinical LV, CL20-i650–MND-WAS. Here, we evaluate the efficacy and safety of the insulted vs uninsulated LV (CL20-MND-WAS) in a large cohort of primary and secondary transplanted Was/- mice. Both vectors enabled stable engraftment of WASp expressing cells in all hematopoietic lineages, positive selection of WASp+ cells, and rescued functional and developmental T and B cell defects without evidence for toxicity. Inclusion of the insulator allowed for higher expression levels at lower numbers of viral integration per cell; a feature that was further exaggerated in secondary recipients. Finally, in parallel, we evaluated CL20-i650–MND LV expressing a fluorescent marker using CD34+ autologous HSC transplants in a non-human primate model (Macaca nemestrina) and observed long term stable polyclonal engraftment of LV expressing hematopoietic cells for up to 2 yr. Based on these extensive pre-clinical and toxicity studies we anticipate that CL20-i650–MND-WAS LV will provide sustained rescue of platelet and lymphocyte development and function in vivo (facilitated, in part, via the insulator) without evidence of toxicity. We plan to directly test this hypothesis in an upcoming WAS LV clinical trial.

24. Targeted Anticancer Approach Through microRNA-Regulated Tumor-Specific RNA Replacement

We have previously presented that trans-splicing ribozymes based on Tetrahymena group I intron could be potent anti-cancer devices through diminishing target RNA level and simultaneously triggering anti-cancer gene activity only in cancer cells expressing the target RNA. Particularly, hTERT-targeting ribozyme was shown to specifically and efficiently retard tumors in various xenograft carcinomatosis nude mouse models. However, concerns for the hTERT-targeting approach include potential side effects to proliferating hTERT+ normal cells. In this study, to improve cancer-specificity of the ribozyme, we developed cancer-specific ribozyme expression construct via microRNA regulation. To circumvent hepatocellular carcinoma (HCC) as target models, we created tissue-specific microRNA-regulated ribozyme system by incorporating perfect antisense sequence against normal hepatocyte-selective miRNA-122a that is down-regulated in HCC to the 3’-UTR region of ribozyme. Adenovirus encoding the modified hTERT-targeting ribozyme specifically and efficiently induced transgene and cytotoxicity in liver cancers through cancer-specific RNA replacement. Moreover, specificity of cancer regression was highly increased without compromising its anti-cancer efficacy in xenograft orthotopic HCC model. Furthermore, to assess the effect of this system in syngeneic model, we constructed mTERT-targeting ribozyme harboring miRNA-122a target sites. Adenoviral vector encoding the modified ribozyme selectively and efficiently regressed mTERT+ HCC with minimal liver toxicity due to least ribozyme expression in the normal livers in syngeneic orthotopic mouse model. Noticeably, cytotoxicity caused by the adenovirus induced specific immunogenic cancer cell death in the syngeneic model, thereby completely suppressing secondary tumor challenge in the mouse. Therefore, specifically targeted transgene induction through microRNA-regulated RNA replacement provides novel strategies for efficient and safe cancer gene therapy.

165. Theophylline-Dependent Aptazyme as a Novel Tool for Transgene Expression Regulation in Mammalian Cells

Successful and safe gene therapy approaches rely on regulatory tools ensuring both specific and controlled expression of the therapeutic gene. To ensure safety, timing or dosing in patients, external control of therapeutic gene expression is desirable or even required. In this study, we explored the potential of artificial aptazymes, ligand-dependent self-cleaving ribozymes, as an innovative tool, for regulation of therapeutic gene expression. For cloning of dual-luciferase reporter plasmid pTA-luc, psiCheck-2 (Promega) was used as template vector. The theophylline-dependent aptazyme was inserted into the 3’-UTR of the Gaussia luciferase gene. Quantification of reporter activity after plasmid transfection of HEK293 cells showed the basal level of less than 10% of psiCheck-2, and dose-dependent turn-on regulation of protein expression by theophylline. The maximum magnitude of the induction versus basal level was ~4-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 1 mM, facilitating the fine-tuning of transgene expression. The similar results came after changing the reporter gene to Kallistatin, but the regulatory fold can be quite different. Actually, in our study, not every aptazyme can be improved in regulatory ability by mutating the upstream start codon from AUG to AAG to avoid nonspecific recognition by the small ribosomal subunit, which may lead to drastic reduction in regulation. However, by inserting aptazyme in different site or using the tandem one, we made it done. The regulatory range of the single insertion system appeared almost 3 times exactly wider than the original system, let alone the ability of the tandem one. And we have evidence to believe that the regulatory ability will be various according to the insertion site and the vector. The aptazyme-based control system was further extended to HeLa and A549 cells, and demonstrated to function in a rapid, reversible and dose-dependent manner, suggesting potential for therapeutic applications in humans. Unfortunately, due to the toxicity of theophlline, the regulatory ability can only be defined in theory, and the decrease of ligand concentration would cause the reduction of regulatory ability. More safer ligands are urgent required in the futrue. View Large Image | Download PowerPoint Slide

Gene therapy to treat cardiac arrhythmias.

Gene therapy to treat electrical dysfunction of the heart is an appealing strategy because of the limited therapeutic options available to manage the most-severe cardiac arrhythmias, such as ventricular tachycardia, ventricular fibrillation, and asystole. However, cardiac genetic manipulation is challenging, given the complex mechanisms underlying arrhythmias. Nevertheless, the growing understanding of the molecular basis of these diseases, and the development of sophisticated vectors and delivery strategies, are providing researchers with adequate means to target specific genes and pathways involved in disorders of heart rhythm. Data from preclinical studies have demonstrated that gene therapy can be successfully used to modify the arrhythmogenic substrate and prevent life-threatening arrhythmias. Therefore, gene therapy might plausibly become a treatment option for patients with difficult-to-manage acquired arrhythmias and for those with inherited arrhythmias. In this Review, we summarize the preclinical studies into gene therapy for acquired and inherited arrhythmias of the atria or ventricles. We also provide an overview of the technical advances in the design of constructs and viral vectors to increase the efficiency and safety of gene therapy and to improve selective delivery to target organs.

Gene therapy for nucleus pulposus regeneration by heme oxygenase-1 plasmid DNA carried by mixed polyplex micelles with thermo-responsive heterogeneous coronas

Safe and high-efficiency gene therapy for nucleus pulposus (NP) regeneration was urgently desired to treat disc degeneration-associated diseases. In this work, an efficient nonviral cationic block copolymer gene delivery system was used to deliver therapeutic plasmid DNA (pDNA), which was prepared via complexation between the mixed cationic block copolymers, poly(ethylene glycol)-block-poly{N-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide} [PEG-b-PAsp(DET)] and poly(N-isopropylacrylamide)-block-PAsp(DET) [PNIPAM-b-PAsp(DET)], and pDNA at 25 °C. The mixed polyplex micelles (MPMs) containing heterogeneous coronas with hydrophobic and hydrophilic microdomains coexisting could be obtained upon heating from 25 to 37 °C, which showed high tolerability against nuclease and strong resistance towards protein adsorption. The gene transfection efficiency of MPMs in NP cells was significantly higher than that of regular polyplex micelles prepared from sole block copolymer of PEG-b-PAsp(DET) (SPMs) in in vitro and in vivo evaluation due to the synergistic effect of improved colloidal stability and low cytotoxicity. High expression of heme oxygenase-1 (HO-1) in NP cells transfected by MPMs loading HO-1 pDNA significantly decreased the expression activity of matrix metalloproteinases 3 (MMP-3) and cyclo-oxygenase-2 (COX-2) induced by interleukin-1β (IL-1β), and simultaneously increased the NP phenotype-associated genes such as aggrecan, type II collagen, and SOX-9. Moreover, the therapeutic effects of MPMs loading pDNA were tested to treat disc degeneration induced by stab injury. The results demonstrated that administration of HO-1 pDNA carried by MPMs in rat tail discs apparently reduced inflammatory responses induced by need stab and increased glycosaminoglycan (GAG) content, finally achieving better therapeutic efficacy as compared with SPMs. Consequently, MPMs loading HO-1 pDNA were demonstrated to be potential as a safe and high-efficiency nonviral gene delivery system for retarding or regenerating the degenerative discs.