Science is host to a constantly emerging series of new paradigms, and it is this characteristic that makes science both interesting and dynamic. As a part of this continuum, it became possible to create recombinant DNA molecules. Immediately it was recognized that there was a potential for serious adverse events associated with this new technology. Following two scientific conferences at Asilomar, California, the National Institutes of Health moved quickly to create the Recombinant DNA Advisory Committee (RAC). For approximately 38 years the RAC has served as an open forum for review of various recombinant DNA experiments, and for the last 23 years it has played a pivotal role in the oversight of human gene therapy. The RAC's existence obviated the need for more restrictive governmental legislation and has supported the development of genetic interventions that are leading to actual human therapies.
In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×105±3.3×105 (MC-Luc) versus 0.4×105±0.3×105 (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31+ expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.
We have examined the role of a novel cytokine, interleukin-27 (IL-27), in mediating interactions between prostate cancer and bone. IL-27 is the most recently characterized member of the family of heterodimeric IL-12-related cytokines and has shown promise in halting tumor growth and mediating tumor regression in several cancer models, including prostate cancer. Prostate cancer is frequently associated with metastases to the bone, where the tumor induces a vicious cycle of communication with osteoblasts and osteoclasts to induce bone lesions, which are a significant cause of pain and skeletal-related events for patients, including a high fracture risk. We describe our findings in the effects of IL-27 gene delivery on prostate cancer cells, osteoblasts, and osteoclasts at different stages of differentiation. We applied the IL-27 gene delivery protocol in vivo utilizing sonoporation (sonodelivery) with the goal of treating and reducing the growth of prostate cancer at a bone metastatic site in vivo. We used a new model of immune-competent prostate adenocarcinoma and characterized the tumor growth reduction, gene expression, and effector cellular profiles. Our results suggest that IL-27 can be effective in reducing tumor growth, can help normalize bone structure, and can promote enhanced accumulation of effector cells in prostate tumors. These results are promising, because they are relevant to developing a novel IL-27-based strategy that can treat both the tumor and the bone, by using this simple and effective sonodelivery method for treating prostate tumor bone metastases.
Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5–58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.
Gene transfer to both cone and rod photoreceptors (PRs) is essential for gene therapy of inherited retinal degenerations that are caused by mutations in genes expressed in both PR types. Vectors based on the adeno-associated virus (AAV) efficiently transduce PRs of different species. However, these are predominantly rods and little is known about the ability of the AAV to transduce cones in combination with rods. Here we show that AAV2/8 transduces pig cones to levels that are similar to AAV2/9, and the outer nuclear layer (mainly rods) to levels that are on average higher, although not statistically significant, than both AAV2/5 and AAV2/9. We additionally found that the ubiquitous cytomegalovirus (CMV), but not the PR-specific GRK1 promoter, transduced pig cones efficiently, presumably because GRK1 is not expressed in pig cones as observed in mice and humans. Indeed, the GRK1 and CMV promoters transduce a similar percentage of murine cones with the CMV reaching the highest expression levels. Consistent with this, the AAV2/8 vectors with either the CMV or the GRK1 promoter restore cone function in a mouse model of Leber congenital amaurosis type 1 (LCA1), supporting the use of AAV2/8 for gene therapy of LCA1 as well as of other retinal diseases requiring gene transfer to both PR types.
Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course.
The naturally occurring adeno-associated virus (AAV) isolates display diverse tissue tropisms in different hosts. Robust cardiac transduction in particular has been reported for certain AAV strains. Successful applications of these AAV strains in preclinical and clinical settings with a focus on treating cardiovascular disease continue to be reported. At the same time, these studies have highlighted challenges such as cross-species variability in AAV tropism, transduction efficiency, and immunity. Continued progress in our understanding of AAV capsid structure and biology has provided the rationale for designing improved vectors that can possibly address these concerns. The current report provides an overview of cardiotropic AAV, existing gaps in our knowledge, and newly engineered AAV strains that are viable candidates for the cardiac gene therapy clinic.
Sonia I. Skarlatos, PhD (September 28, 1953–August 6, 2013), was the deputy director of the Division of Cardiovascular Sciences at the National Heart, Lung, and Blood Institute (NHLBI). This article reviews her work in establishing, leading, or facilitating extramural translational research programs supported by the NHLBI, specifically focusing on her work as a consistent advocate for the advancement of gene and cell therapies.
Metabolic myopathies are a diverse group of rare diseases in which impaired breakdown of stored energy leads to profound muscle dysfunction ranging from exercise intolerance to severe muscle wasting. Metabolic myopathies are largely caused by functional deficiency of a single gene and are generally subcategorized into three major types of metabolic disease: mitochondrial, lipid, or glycogen. Treatment varies greatly depending on the biochemical nature of the disease, and unfortunately no definitive treatments exist for metabolic myopathy. Since this group of diseases is inherited, gene therapy is being explored as an approach to personalized medical treatment. Adeno-associated virus–based vectors in particular have shown to be promising in the treatment of several forms of metabolic myopathy. This review will discuss the most recent advances in gene therapy efforts for the treatment of metabolic myopathies.
Immune cells are involved in the pathogenesis of osteoarthritis (OA). CD4+ T cells were activated during the onset of OA and induced macrophage inflammatory protein (MIP)-1γ expression and subsequent osteoclast formation. We evaluated the effects of local knockdown of MIP-1γ in a mouse OA model induced by anterior cruciate ligament transection. The mouse macrophage cell lines and osteoclast-like cells generated from immature hematopoietic monocyte/macrophage progenitors of murine bone marrow were cocultured with either receptor activator of NFκB ligand (RANKL) or CD4+ T cells. The levels of MIP-1γ and RANKL in cells and mice were examined by enzyme-linked immunosorbent assay (ELISA). The osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase and cathepsin K staining. OA was induced in one hind-leg knee joint of B6 mice. Lentiviral vector encoding MIP-1γ small hairpin RNA (shRNA) and control vector were individually injected intra-articularly into the knee joints, which were histologically assessed for manifestations of OA. The expression of MIP-1γ and matrix metalloproteinase (MMP)-13 and the infiltration of CD4+ T cells, macrophages, and osteoclastogenesis in tissues were examined using immunohistochemistry. CD4+ T cells were involved in OA by inducing MIP-1γ expression in osteoclast progenitors and the subsequent osteoclast formation. Neutralizing MIP-1γ with a specific antibody abolishes RANKL-stimulated and CD4+ T-cell-stimulated osteoclast formation. MIP-1γ levels were significantly higher in synovium and the chondro-osseous junction of joints 90 days postsurgery. The number of infiltrated CD4+ T cells and macrophages and IL-1β expression were reduced in the synovial tissues of mice treated with MIP-1γ shRNA. Histopathological examinations revealed that mice treated with MIP-1γ shRNA had less severe OA than control mice had, as well as decreased osteoclast formation and MMP-13 expression. Locally inhibiting MIP-1γ expression may ameliorate disease progression and provide a new OA therapy.
Shen and colleagues reveal that CD4+ T cells are involved in osteoarthritis via induction of MIP-1γ in osteoclast progenitors, which leads to osteoclast formation. They demonstrate that shRNA-mediated knockdown of MIP-1γ leads to decreased osteoclast formation and reduced osteoarthritis in mice.
Successful stem cell gene therapy requires high numbers of genetically engineered hematopoietic stem cells collected using optimal mobilization strategies. Here we focus on stem cell mobilization strategies for thalassemia and present the results of a plerixafor-based mobilization trial with emphasis on the remobilization with granulocyte-colony stimulating factor (G-CSF)+plerixafor in those patients who had previously failed mobilization. Plerixafor rapidly mobilized CD34+ cells without inducing hyperleukocytosis; however, 35% of patients failed to reach the target cell dose of ≥6×106 CD34+ cells/kg. Four subjects who failed on either plerixafor or G-CSF were remobilized with G-CSF+plerixafor. The combination proved highly synergistic; the target cell dose was readily reached and the per-apheresis yield was significantly increased over initial mobilization, ultimately resulting in single-apheresis collections, despite a more than 50% reduction of the dose of G-CSF in splenectomized patients to avoid hyperleukocytosis. The total stem and progenitor cells mobilized in G-CSF+plerixafor patients were higher than in patients treated by plerixafor alone. Importantly, the G-CSF+plerixafor-mobilized cells displayed a primitive stem cell phenotype and higher clonogenic capacity over plerixafor-mobilized cells. G-CSF+plerixafor represents the optimal strategy when very high yields of stem cells or a single apheresis is required. The high yields and the favorable transplantation features render the G-CSF+plerixafor-mobilized cells the optimal CD34+ cell source for stem cell gene therapy applications.
Yannaki and colleagues evaluate methods for mobilizing hematopoietic stem cells (HSCs) in patients with thalassemia major. They identify a combination regimen of granulocyte-colony stimulating factor (GCSF) and Plerixafor that circumvents the toxicity of GCSF previously reported in thalassemia patients while also providing high HSC yields in a single apheresis from patients who previously failed single agent regimens. The excellent yield and clonogenic capacity of the mobilized HSCs suggests that this method may be broadly useful for ex vivo HSC gene transfer protocols.
Mesenchymal stem cell (MSC) therapy offers the potential to promote recovery after myocardial infarction (MI). However, therapeutic efficacy may be limited by poor survival and retention of transplanted cells. A combination of gene and cell therapy has the capacity to prevent donor cell death and augment the reparative and regenerative effects of cell transfer. The present study investigates the effect of exogenous heat shock protein 27 (Hsp27) expression in MSCs in an in vitro model of ischemia and in an in vivo rat MI model and aims to determine if this could enhance the therapeutic benefit associated with cell delivery. Hsp27 overexpression by lentivirus vector modification resulted in increased MSC survival in vitro and in vivo. Furthermore, decreased apoptosis in the infarcted tissue and improved cardiac function was observed in the Hsp27 group, enhancing the therapeutic effect of MSCs. Together, these data demonstrate that ex vivo genetic modification—specifically Hsp27 overexpression—offers the possibility of enhancing the efficacy of MSC therapy in MI.
McGinley and colleagues demonstrate that mesenchymal stem cells (MSCs) can be rendered resistant to ischemia and hypoxia in vitro through lentivirus-mediated overexpression of heat shock protein 27 (Hsp27). In a rat model of myocardial ischemia, Hsp27-modified MSCs exhibit improved engraftment compared with unmodified cells with associated benefits in cardiac function.
Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1–2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity.
De Oliveira and colleagues present a method to continuously generate effector cells targeting B cell neoplasms in vivo by modifying human hematopoietic stem/progenitor cells (HSPCs) to carry a CD19-specific chimeric antigen receptor (CAR). The modified HSCs produce effector cells of multiple lineages with cytolytic activity against CD19-expressing cells.
A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.
Zhong and colleagues use a nested PCR assay to examine whether adenoviral gene transfer led to the development of cancer in two clinical research subjects with ornithine transcarbamylase deficiency. Using this approach, they were unable to detect adenovirus vector genomes in normal or tumor tissues from the subjects more than a decade after vector infusion. These results suggest that adenoviral gene transfer was an unlikely contributor to the colon cancer or hepatocellular carcinoma observed in the two research subjects.
We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.
Yan and colleagues identify a protease-sensitive factor in ferret airway secretions that profoundly reduces transduction by AAV1. The unidentified factor inhibits both vector entry and postentry processing in polarized ferret airway epithelial (FAE) cell cultures. Transduction can be rescued both in vitro and in vivo by a proteasome inhibitor, suggesting this novel factor interacts with the ubiquitin–proteasome system during intracellular virus processing.
Duchenne muscular dystrophy (DMD) typically occurs as a result of truncating mutations in the DMD gene that result in a lack of expression of the dystrophin protein in muscle fibers. Various therapies under development are directed toward restoring dystrophin expression at the subsarcolemmal membrane, including gene transfer. In a trial of intramuscular adeno-associated virus (AAV)-mediated delivery of a therapeutic minidystrophin construct, we identified in two of six subjects the presence of a population of T cells that had been primed to recognize dystrophin epitopes before transgene delivery. As the presence of preexisting T cell immunity may have a significant effect on the success of therapeutic approaches for restoring dystrophin, we sought to determine the prevalence of such immunity within a DMD cohort from our Muscular Dystrophy Association clinic. Dystrophin-specific T cell immunity was evaluated in subjects with DMD who were either receiving the glucocorticoid steroid prednisone (n=24) or deflazacort (n=29), or who were not receiving steroids (n=17), as well as from normal age-matched control subjects (n=21). We demonstrate that increasing age correlates with an increased risk for the presence of anti-dystrophin T cell immunity, and that treatment with either corticosteroid decreases risk compared with no treatment, suggesting that steroid therapy in part may derive some of its benefit through modulation of T cell responses. The frequency of dystrophin-specific T cells detected by enzyme-linked immunospot assay was lower in subjects treated with deflazacort versus prednisone, despite similar overall corticosteroid exposure, suggesting that the effects of the two corticosteroids may not be identical in patients with DMD. T cells targeted epitopes upstream and downstream of the dystrophin gene mutation and involved the CD4+ helper and/or CD8+ cytotoxic subsets. Our data confirm the presence of preexisting circulating T cell immunity to dystrophin in a sizable proportion of patients with DMD, and emphasize the need to consider this in the design and interpretation of clinical gene therapy trials.
Flanigan and colleagues characterize the prevalence of preexisting dystrophin-specific T cells in Duchenne muscular dystrophy (DMD) patients. They identify CD4+ and CD8+ T cell populations targeting epitopes upstream and downstream of dystrophin mutations in a significant fraction of patients. They further demonstrate a lower frequency of dystrophin-specific T cells in patients receiving glucocorticoid therapy. These findings suggest important considerations for future DMD gene therapy trials and offer new insight into the mechanism of glucocorticoid therapy for DMD.
Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5×1011 genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV −dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca2+ transient studies showed an increased rate of Ca2+ decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.
Gao and colleagues utilize systemic adeno-associated virus–mediated gene delivery to achieve stable serum concentrations of urocortin 2 (UCn2), a peptide with demonstrated capacity to support cardiac function in congestive heart failure but limited utility due to an extremely short half-life. Mice treated with the UCn2-encoding vector exhibit sustained supraphysiologic expression of UCn2 and enhanced left ventricular function for up to 7 months.