Scholarly article on topic '42. Uniform Scale-Independent Gene Transfer to Striated Muscle after Transvenular Extravasation of Vector'

42. Uniform Scale-Independent Gene Transfer to Striated Muscle after Transvenular Extravasation of Vector Academic research paper on "Basic medicine"

Share paper
Academic journal
Mol Ther
OECD Field of science

Academic research paper on topic "42. Uniform Scale-Independent Gene Transfer to Striated Muscle after Transvenular Extravasation of Vector"

40. Enhanced Factor IX Delivery from Bioengineered Hybrid Human Skeletal Muscle Co-Expressing VEGF

Lieven Thorrez,1 Herman Vandenburgh,1,2 Desire Collen,1 Janet Shansky,2 Thierry VandenDriessche,1 Marinee Chuah.1 'Center for Transgene Technology and Gene Therapy, University of Leuven KUL/Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium; 2Department of Pathology, Brown Medical School/Miriam Hospital, Providence, RI.

The continuous development of efficient and safe gene delivery approaches is a prerogative to move the field of gene therapy forward. We have recently been focusing on the development of novel gene delivery approaches based on the use of genetically engineered adult human stem cells to establish a novel ex vivo gene therapy paradigm at the cross-roads of gene therapy, stem cell technology and tissue engineering. Adult human skeletal muscle stem cells are easy to obtain by needle biopsy and can be efficiently transduced ex vivo with third generation lentiviral vectors. With GFP as a marker gene, nearly 100% of cells were shown to be transduced and using the human blood clotting factor IX (FIX) as a transgene, unprecedented levels (>5 |g /106 cells/day) of FIX were obtained in vitro, whereas only 0.4 |g rhFIX/106 cells/day was secreted when retroviral vectors were employed. These cells can be efficiently bioengineered in vitro into postmitotic muscle fibers and implanted subcutaneously as bioartificial muscles (BAMs). The bio-engineered tissues can be removed in the event of an adverse reaction, which significantly improves the safety margin of this technology and which distinguishes this potential "reversible" gene therapy approach from most other gene therapy strategies. Implantation of BAMs transduced ex vivo with lentiviral-GFP vectors resulted in robust and long-term GFP expression in NOD-SCID mice. FIX secreting BAMs implanted subcutaneously into NOD-SCID mice, secreted FIX into the circulation for greater than 90 days but the plasma levels were below therapeutic levels after 20 days. When the muscle fibers were bioengineered from human GFP myoblasts and human myoblasts genetically transduced to express vascular endothelial growth factor (VEGF), significantly more fluorescence was detected after 1 month compared to BAMs without VEGF secretion. A network of new blood vessels was established around the hybrid GFP-VEGF BAMs within 1 month, concomitant with a localized increased vascular permeability around these hybrid BAMs. Using hybrid FIX-VEGF BAMs, the circulating levels of FIX increased significantly and plasma levels of FIX were maintained above therapeutic levels long term. The present study suggests that a limited angiogenic response may contribute to the inability to obtain long-term transgene expression levels following ex vivo gene therapy, as previously shown in a phase I/II clinical trial for hemophilia (Roth et al., N Engl J Med. 344:1735, 2001). This study provides novel insights into the mechanisms that improve the outcome of tissue engineering and gene therapy, particularly by modulating angiogenesis. Implantable tissues bioengineered from genetically engineered muscle cells may provide an alternative and safer approach to in vivo gene transfer for chronic protein delivery. To our knowledge, this is the first demonstration that adult human stem cells transduced with lentiviral vectors can give rise to prolonged circulating clotting factor levels in vivo.

Cardiac Gene Transfer

41. High Efficiency, Catheter-Based, Gene Transfer to the Large Animal Heart

David M. Kaye,1 Kenneth Chien,2 Masahiko Hoshijima,2 Krisztina Zsebo,3 John Power.1,4

'Cardiac Division, Baker Heart Research Institute, Melbourne, Australia; 2Institute for Molecular Medicine, UCSD, La Jolla, CA; 3Celladon Corporation, La Jolla, CA; 4V-Kardia Pty Ltd, Melbourne, VIC, Australia.

Congestive heart failure (HF) is a leading cause of hospitalization, disability and death. Despite significant advances in pharmacotherapy, HF remains a progressive disorder with an unacceptably high mortality rate. While the molecular and cellular basis of the impairment of myocardial contractility has been determined in detail, current beneficial therapies for heart failure only act indirectly, by interfering with neurohormonal control of the heart. Accordingly, considerable attention has recently been directed at the identification of appropriate targets for gene therapy. In particular, studies directed at the excitation-contraction pathway of the heart have identified genes (particularly phospholamban and SERCA) that can be manipulated to improve contractility. As such, translation into clinical practice now requires the development of safe, minimally invasive methods that provide homogeneous delivery to the heart.

We have developed a novel clinically-relevant, percutaneous system for the delivery of viral vectors to the myocardium, based upon an antegrade coronary arterial delivery system combined with recirculation of the coronary venous effluent via a membrane-pump oxygenator circuit. We have demonstrated the safe, reproducible, delivery of adeno-LacZ to the myocardium of both healthy normal sheep (n=15) and sheep with moderate to severe pacing induced heart failure (n=7). During 10-15 minutes recirculation, there was no evidence of compromise in myocardial function (as assessed by echocardiography and electrocardiographic monitoring). Similarly, there was no evidence of lactic acid accumulation in the recirculating blood (baseline vs 15 mins: 2.4 mmol/L vs 2.5 mmol/L). A homogeneous pattern of LacZ delivery was apparent by immunohistochemistry, 2 weeks after gene transfer and no evidence of tissue damage was evident on H+E staining. Of additional importance this 'closed-loop' recirculation system results in minimal systemic leakage (brain, kidney, liver and lung) of viral vector.

DK, JP are founders of V-Kardia. KC is a founder of Celladon. KZ is an employee of Celladon.

42. Uniform Scale-Independent Gene Transfer to Striated Muscle after Transvenular Extravasation of Vector

Kapil Gopal,1 Leonard T. Su,1 Zhonglin Wang,1 Xiaoqing Yin,1 Anthony Nelson,1 Benjamin W. Kozyak,1 James M. Burkman,1 Marilyn A. Mitchell,1 David W. Low,1 Charles R. Bridges,1 Hansell H. Stedman.1,2

'Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA; 2Department of Cell and Molecular Biology, University of Pennsylvania School of Medicine, Philadelphia, PA.

The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for full complementation of the underlying genetic disorder. As a direct approach to this clinical challenge, somatic gene transfer will require efficient, scale-independent transport of DNA-containing

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright © The American Society of Gene Therapy

macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions. We tested the hypothesis that rapid, mechanical distention of the post-capillary venular endothelium by afferent infusion from a distal site would safely facilitate macromolecular transport from the vascular space to the striated muscle interstitium. We show that pressurized infusion through a large-bore catheters in either peripheral, superficial veins or the coronary sinus results in uniform, scale- and vector-independent transduction of myofibers in anatomic domains isolated from the remainder of the circulation. This approach is rapid, minimally invasive as applied to the isolated limb, and avoids pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to virtually 100% of the muscle fibers of an entire extremity in the dog, providing a firm foundation for studies of efficacy in canine models for human diseases. Additional data from a combination of angiographic, tracer dye, and marker gene studies suggests that this approach can be modified to meet the requirements for cardiac-specific or systemic gene delivery as appropriate in a variety of inherited and acquired diseases including hemophilia, muscular dystrophy, and cardiomyopathy.

Figure 1 - p-galactosidase levels of rat limb, rat cardiac and dog limb muscles after no treatment, vector delivery without afferent transvenular retrograde extravasation (ATVRX) and vector delivery with ATVRX.

Figure 2 - LacZ expression after rat quadriceps (left), rat heart (middle), and dog vastus medialis (right) stained with x-galactosidase.

43. Targeting the Biology of Heart Disease: Engineered Zinc Finger Protein Repressors of Phospholamban as a Potential Therapy for Congestive Heart Failure

H. Steve Zhang,1 Lei Zhang,1 Yan Huang,2 Dinggang Liu,2 Yuxin Liang,1 Reed Hickey,2 Dmitry Guschin,1 Simon Chandler,1 Mike Kunis,1 Linda Hinh,1 Danny Xia,1 Xiaohong Zhong,1 S. Kaye Spratt,1 J. Tyler Martin,1 Casey C. Case,1 Dale Ando,1 Edward J. Rebar,1 Philip D. Gregory,1 Frank Giordano.2 1Dept. of Therapeutic Gene Regulation, Sangamo BioSciences Inc., Richmond, CA; 2Dept. of Medicine, Yale University School of Medicine, New Haven, CT.

Improper calcium handling of the heart is a hallmark of patients with congestive heart failure (CHF). Because calcium is critical for cardiac contractility, proteins that regulate calcium homeostasis are potential targets treating CHF. Phospholamban (PLN) decreases contractility by inhibiting the activity of Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a); an increased PLN/SERCA2a ratio is often found in CHF patients. Recent studies have

demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of CHF, suggesting that down-regulation of PLN may improve cardiac function in CHF patients. Importantly, inhibition of PLN enhances calcium handling without activating b-adrenergic pathways, which is known to have many side-effects and increase mortality. The development of small-molecule inhibitors of PLN function has so far been unsuccessful, largely due to the difficulty of inhibiting protein-protein interactions (such as that between PLN and SERCA2a) using small molecules. On the other hand, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect.

As part of a therapeutic program in CHF, we have engineered zinc finger protein (ZFP) transcriptional repressors that target either the human or rat PLN promoter. The rat-specific ZFP repressor gave >90% reduction of PLN mRNA in a rat heart-derived cell line; and the human-specific ZFP produced a similar level of repression in human smooth muscle cells. Microarray analyses indicated that both the rat- and human-targeted ZFPs operated with exquisite specificity, with PLN being the only gene that was significantly repressed within the monitored genome. When the rat PLN repressor was introduced into primary cardiomyocytes of neonatal rats, it efficiently repressed PLN transcription, despite the high level of PLN expression in these cells. Furthermore, when the same ZFP was introduced into adult rat hearts, subsequently isolated ZFP-positive myocytes showed accelerated calcium transients as well as improved contractility, highlighting the functional significance of ZFP mediated PLN repression. Moreover, initial data from a rat model of CHF in which the PLN repressing ZFP TF was delivered to the myocardium using adeno-associated virus (AAV)-based vectors indicated improvements in several hemodynamic parameters consistent with improved heart function post-treatment. These data support further investigation of delivery modes and vectors in additional rat models of heart failure to provide formal pre-clinical validation of these promising reagents.

44. Development of AAV-Mediated Gene Therapy for Murine Models of Genetic Diseases Affecting the Heart

Christina A. Pacak,1,2 Cathryn Mah,2,4 Gabriel Gaidosh,3 Melissa Lewis,2 Raquel Torres, Kevin Campbell,5 Glenn A. Walter,3 Barry J. Byrne.124

Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 2Powell Gene Therapy Center, University of Florida, Gainesville, FL; 3Physiology, University of Florida, Gainesville, FL; 4Cellular and Molecular Therapy, University of Florida, Gainesville, FL; 5HHMI, University of Iowa, Iowa City, IA.

The long term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of genetic diseases affecting the heart. Due to its small size, safety and proven ability to persist for long periods of time in muscle, adeno-associated virus (AAV) has emerged as a promising cardiac gene delivery vehicle. We have sought to determine the most advantageous combination AAV serotype and vector delivery route for the transduction of cardiomyocytes in vivo. Both intra-venous (iv) and intra-cardiac (ic) injection routes were compared by injecting 1x1011 and 5x1010 (respectively) vector particles of AAV-CMV-LacZ per mouse neonate of 3 different serotypes AAV1, AAV8 and AAV9. Tissue analysis included both x-gal staining on tissue sections to visualize expression and the quantitative p-galactosidase enzyme detection assay. Our results show that iv administration of AAV9 results in 30-fold more efficient transduction of cardiac tissue than AAV1. Moreover, hearts injected with AAV9 displayed a global distribution of transgene expression suggesting this serotype has no transduction site

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright © The American Society of Gene Therapy