Safety and feasibility of transvenous limb perfusion with normal saline in human muscular dystrophy.
Due to their genetic basis and the current lack of curative therapy, the muscular dystrophies are excellent candidate diseases for gene therapies. An essential step in this development of such therapies is delivery of genetic material to a single limb to demonstrate safety and efficacy prior to systemic administration. Of the various methods of single limb delivery studied in experimental animals, high-pressure, high-volume transvenous limb perfusion shows the greatest potential to be an effective, clinically practical and generally applicable approach. In this project, we will perform a safety and feasibility study of transvenous single limb perfusion with normal saline in human subjects with muscular dystrophy. The study is designed as a dose escalation safety study with the perfusion parameters increased in a stepwise manner and careful monitoring for both local and systemic toxicity. This design will also permit us to address the multiple logistical aspects inherent in going from animals to humans with muscular dystrophy including analgesia, vascular access and larger infusion volumes. In Specific Aim 1, we will study young adults with limb-girdle or Becker muscular dystrophy beginning with infusions of .05 ml saline/ml of limb volume and escalating to a maximum of 0.4 ml/ml. An independent safety monitor will review data on each subject (perfusion parameters, laboratory and clinical testing) and must approve planed perfusion parameters for the next subject. From this study, we will determine the maximum perfusion parameters that are safe and document the degree of fluid delivery into muscle by T2 MRI. In Specific Aim 2, we will carry out a similar dose escalation study in adults with Duchenne muscular dystrophy beginning at .05 ml saline/ml of limb volume and escalating to the maximum volume determined from Specific Aim 1 as posing no greater than minimal risk. This study will provide the necessary safety and feasibility data on the perfusion technique itself to provide the basis for future regional limb delivery studies of active gene therapy. The results will be generally applicable to the single limb delivery of many different agents (oligonucleotides, plasmid -DNA and viral delivery systems) and to patients of different ages with diverse types of muscular dystrophy.
Evaluate hydrodynamic limb vein (HLV) and isolated limb artery (ILA) injection methods for delivery of various AAV minidys vectors in a dog model of muscular dystrophy.
This project will focus on the DMD dog model (GRMD) to investigate blood-vessel-mediated gene delivery of AAV vectors into the limbs and eventually the whole body. Specific Aim 1 is designed to compare regional gene delivery efficiency of arterial and venous methods in the limbs. Arterial delivery with pressure and/or with vessel dilators was investigated in early studies by a few labs. But more recent studies have switched to the pressurized intravenous delivery methods. However, a latest report revived the arterial method again, but without the use of any additional pressure and drugs. These methods have their pros and cons, but are never compared side-by-side. In Aim 1 we propose to compare and optimize the regional AAV vector delivery methods in dog limbs. Specific Aim 2 is designed to examine systemic gene delivery efficiency of AAV8 and AAV9 in dogs. Previously, we and others have shown that new serotypes of AAV can achieve whole body gene delivery by simple intravenous injection in mice and hamsters. In Aim 2, we plan to explore both AAV8 and AAV9 for systemic gene delivery in the dogs. We will also examine any novel AAV vectors yielded by Project 2.
AAV vectors for targeted and repeated delivery
This project will capitalize on AAV vector results derived from the ongoing AAV Phase I DMD clinical trial. The advent of novel AAV vectors (serotypes 1-11) and the solving of the capsid crystal structure has allowed us and others to carry out rational design of AAV capsids to generate laboratory strains of gene delivery vehicles. Using such an approach, we have been successful in engineering AAV type 2 liver tropic vectors into a muscle tropic virus using only 5 amino acids derived from the AAV type 1 capsid backbone. This novel chimeric reagent (AAV 2.5) has a distinct immune profile when compared to capsid backbone of parent serotypes and has high efficiency for muscle transduction. Due to the advantages of this chimeric AAV particle we recently initiated a Phase I gene therapy clinical trial for DMD with Dr. Xiao. Early information derived from these efforts has provided hypothesis driven approaches to better understand basic steps such as immune response to viral capsid, viral-receptor interaction, intracellular trafficking and trangene expression. In the proposed studies, we hope to capitalize on these observations by deriving a collection of disease tropic vectors with novel immune profile that will allow for repeat administration. These objectives will be carried out by availability to AAV DNA capsid library technology, selection against patient neutralizing serum (including ongoing AAV clinical trial) and use of large animal models carrying muscle specific disease traits (Project 3). Our objective is to better understand the molecular mechanism of efficient vector muscle transduction that will allow these and other novel delivery reagents for skeletal and heart specific muscle gene therapy. Success of these studies will facilitate the immediate goals of Project 2 and the long-term objective of initiating clinical trials for muscle disorders.
Specific aim 1: generate AAV2.5-derived Nab escape mutants to enable repeat administration in clinical trials
Specific aim 2: Generate 2nd generation AAV vectors capable of evading pre-existing human NAbs and achieving global transduction following systemic administration in the canine DMD model.
