SBIR-STTR Award

AtheroSclerotic CardioVascular Disease (ASCVD) is caused by inflammation within arteries. White blood cells enter the arterial walls, macrophages become engorged with lipid/cholesterol and summon other inflammatory cells to form plaques which ultimately rupture or occlude the artery, killing down-stream tissue (e.g. heart attack). ASCVD remains a major cause of morbidity and mortality of the elderly. Coronary artery disease costs the United States $108.9 billion every year. Strokes and peripheral vascular disease add $30 billion more. World-wide estimates surpass $2 trillion/yr. Thus, there is a large market and a high need. More specifically, we will focus on familial hypercholesterolemia (FH), possibly the most common genetic disease in humans (29219151), or 0.2-0.5% of US population. Those with FH have a coronary mortality dramatically higher than the general population. As ASCVD takes years/decades to develop, adeno-associated virus (AAV)-based gene therapy, documented to last at least 10 years in patients, is an ideal vector for treating FH and ASCVD. AAV has been used in 80 clinical trials world-wide. In this Fast Track proposal, we will further develop a new treatment for FH-ASCVD which includes a new AAV capsid, a new powerful therapeutic gene, fork-head box protein 3 (FOXP3), and a disease-limited transcriptional promoter to express FOXP3 only where needed. Earlier, we used Interleukin (IL)-10 as our therapeutic, “gold standard”, anti-atherosclerosis gene. In subsequent studies, we found FOXP3, the master gene of regulatory T cells (CD4+/CD25+), gives more robust efficacy. AAV8 based gene delivery of FOXP3 into low density lipoprotein receptor knockout (LDLR KO) mice put on high cholesterol diet (HCD) resulted in full inhibition of ASCVD aortic lesions by FOXP3. Yet, indiscriminate overexpression of powerful immunomodulatory genes such as IL10 or FOXP3 may be associated with adverse reactions, in particular increased infections, and even cancer. For the safest design of gene therapy vectors carrying such powerful genes a “disease-specific promoter” has high appeal. A disease-specific promoter will limit expression and serve as a built-in safeguard, yet provide adequate expression at the site of disease to give treatment. The LOX1 gene is known to be transcriptionally up-regulated early in ASCVD/inflammation in a number of cell types, including lymphocytes (lymphoid) and macrophages (myeloid). LOX1 expression in endothelial cells also predates and predicts the sites of future ASCVD. Earlier we tested this disease-limited gene therapy hypothesis by studying an adeno-associated virus vector (AAV2 backbone), using the AAV8 capsid, and containing the full length LOX1 promoter (LOX1pr; 2.4 kb) driving expression of the human (h)IL10, for their anti- atherosclerotic effect in LDLR KO mice on HCD. We compared the AAV2/8.LOX1pr-hIL10 (Kcardio-1™), with the LOX1pr driving hIL10 expression, to AAV2/8.CMVpr-hIL10. The CMVpr is a strong constitutive promoter used for comparison (positive control). The LOX1pr vector gave statistically equal efficacy to the CMVpr vector in down-regulating atherogenesis by IL10. So, in this FastTrack proposal we will use a superior FOXP3 therapeutic cDNA , in place of IL10, and express it from the safe, disease-specific LOX1pr. Now our goal is to generate an AAV2-LOX1pr-FOXP3 vector, with built-in safeguard (LOX1pr) and to test its efficacy in mice and its safety in large animal models. In this FAST TRACK proposal we intend to generate two additional improvements in the AAV 8 capsid proteins (tyrosine substitutions (aa 447 and 733) and ICAM2 aa21-42 peptide insertion [binds CD11a] to the above proven AAV/FOXP3 therapeutic technology (Phase I) generating a more effective AAV2/8(cvd2) LOX1pr- FOXP3 vector (aka Kcardio-2™, product name) for transducing lymphocytes and macrophages. Our Kcardio- 2 will also be tested in isolated human leukocytes (T cells and Mac) for improved gene delivery into these cells, giving increased FOXP3+ phenotype (eg. secretion of IL-10 and TGFbeta1), more Treg, and for efficacy against HCD-induced atherosclerosis in LDLR KO mice. Then, we will test this improved Kcardio-2 vector in toxicology studies in large animal models (Phase II). At the completion of this project we will be ready to launch our AAV2/8(cvd2).LOX1pr-FOXP3 into Phase I and II clinical trials in people with FH that are not controlled with current therapy and predisposed to ASCVD. Moreover, there will be payoffs for this technology across medicine. Most, if not all, diseases of aging have inflammation as a major etiologies (arthritis, Alzheimers and Parkinsons, etc.). Thus the agents we develop here could have broad use across a wide range of diseases, not only ASCVD.

Public Health Relevance Statement:
Cardiovascular disease remains a major cause of morbidity and mortality for Americans, particularly for the 0.2-0.5% of the population with familial hypercholesterolemia (FH) who have many fold higher coronary mortality. Even in those with FH, ASCVD takes decades to develop, and adeno-associated virus (AAV)-based gene therapy, documented to last at least 10 years in patients, is an ideal vector for treating/preventing ASCVD. In this fast track proposal we will generate a modified AAV vector carrying the powerful anti- inflammatory human (h)FOXP3 gene, but which only expresses hFOXP3 only at the site of disease. We will determine the level of AAV/FOXP3 virus need for efficacy against FH-ASCVD in a mouse model (Phase I), and then test for safety, toxicology, in large animals models (Cynomolgus monkeys and duroc piglets). Once passing the milestones, the Kcardio-2 product, AAV/LOX1pr-FOXP3 vector will be ready for Phase I and II clinical trials.

Project Terms:
adeno-associated viral vector; Adverse effects; Adverse reactions; Affect; Aging; Alzheimer's Disease; American; Animal Model; Animals; Anti-inflammatory; Antiatherogenic; Aortic Diseases; Area; Arteries; Arteriosclerosis; Arthritis; atherogenesis; Atherosclerosis; Automobile Driving; base; Binding; Blood; Blood Vessels; Capsid; Capsid Proteins; Cardiovascular Diseases; CD4 Positive T Lymphocytes; Cell Count; cell type; Cells; Cholesterol; Clinical Trials; Complementary DNA; Coronary; Coronary Arteriosclerosis; Coronary heart disease; Cost of Illness; Deltastab; Dependovirus; design; Development; Diet; Disease; Elderly; Endothelial Cells; Engineering; Etiology; Familial Hypercholesterolemia; Family suidae; FOXP3 gene; Future; Gene Delivery; gene delivery system; Gene Expression; gene therapy; Gene Transduction Agent; General Population; Genes; Genetic Diseases; Genetic Transcription; Goals; Gold; Head; Human; ICAM2 gene; IL2RA gene; immunoregulation; Immunosuppression; improved; In Vitro; in vivo; Infection; Inflammation; Inflammatory; Interleukin-10; Knockout Mice; LacZ Genes; Lectin; Length; Lesion; Leukocytes; Lipids; Lipoprotein Receptor; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Lymphocyte; Lymphoid; Macaca fascicularis; macrophage; Malignant Neoplasms; Measures; Medicine; Modification; Monitor; Monkeys; Morbidity - disease rate; mortality; mouse model; Mus; Myelogenous; Myocardial Infarction; Names; novel strategies; overexpression; oxidized low density lipoprotein; Parkinson Disease; Pathogenesis; Patients; Peptides; Peripheral Vascular Diseases; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Phenotype; Play; Population; prevent; Process; promoter; Proteins; Regulator Genes; Regulatory T-Lymphocyte; Role; Rupture; Safety; safety testing; Site; Stroke; T-Lymphocyte; Technology; Testing; Therapeutic; therapeutic gene; Thick; Tissues; Toxicology; transcription factor; transduction efficiency; Tropism; Tyrosine; United States; vector; Vertebral column;