Development and non-clinical GLP testing of a new anti-arteriosclerosis gene therapy delivered by engineered adeno-associated viral vectors.

AtheroSclerotic CardioVascular DiseaseASCVDis caused by inflammation within arteriesWhite blood
cells enter the arterial wallsmacrophages become engorged with lipid cholesterol and summon other
inflammatory cells to form plaques which ultimately rupture or occlude the arterykilling down stream tissuee gheart attackASCVD remains a major cause of morbidity and mortality of the elderlyCoronary artery disease
costs the United States $billion every yearStrokes and peripheral vascular disease add $billion moreWorld wide estimates surpass $trillion yrThusthere is a large market and a high needMore specificallywe
will focus on familial hypercholesterolemiaFHpossibly the most common genetic disease in humansorof US populationThose with FH have a coronary mortality dramatically higher than the
general populationAs ASCVD takes years decades to developadeno associated virusAAVbased gene
therapydocumented to last at leastyears in patientsis an ideal vector for treating FH and ASCVDAAV has
been used inclinical trials world wideIn this Fast Track proposalwe will further develop a new treatment for FH ASCVD which includes a
new AAV capsida new powerful therapeutic genefork head box proteinFOXPand a disease limited
transcriptional promoter to express FOXPonly where neededEarlierwe used InterleukinILas our
therapeuticgold standardanti atherosclerosis geneIn subsequent studieswe found FOXPthe master
gene of regulatory T cellsCDCDgives more robust efficacyAAVbased gene delivery of FOXPinto
low density lipoprotein receptor knockoutLDLR KOmice put on high cholesterol dietHCDresulted in full
inhibition of ASCVD aortic lesions by FOXPYetindiscriminate overexpression of powerful immunomodulatory
genes such as ILor FOXPmay be associated with adverse reactionsin particular increased infectionsand
even cancerFor the safest design of gene therapy vectors carrying such powerful genes adisease specific
promoterhas high appealA disease specific promoter will limit expression and serve as a built in safeguardyet provide adequate expression at the site of disease to give treatmentThe LOXgene is known to be transcriptionally up regulated early in ASCVD inflammation in a number
of cell typesincluding lymphocyteslymphoidand macrophagesmyeloidLOXexpression in endothelial
cells also predates and predicts the sites of future ASCVDEarlier we tested this disease limited gene therapy
hypothesis by studying an adeno associated virus vectorAAVbackboneusing the AAVcapsidand
containing the full length LOXpromoterLOX prkbdriving expression of the humanh ILfor their antiatherosclerotic effect in LDLR KO mice on HCDWe compared the AAVLOX pr hILKcardiowith
the LOX pr driving hILexpressionto AAVCMVpr hILThe CMVpr is a strong constitutive promoter
used for comparisonpositive controlThe LOX pr vector gave statistically equal efficacy to the CMVpr vector
in down regulating atherogenesis by ILSoin this FastTrack proposal we will use a superior FOXPtherapeutic cDNAin place of ILand express it from the safedisease specific LOX prNow our goal is to
generate an AAVLOX pr FOXPvectorwith built in safeguardLOX prand to test its efficacy in mice and
its safety in large animal modelsIn this FAST TRACK proposal we intend to generate two additional improvements in the AAVcapsid
proteinstyrosine substitutionsaaandand ICAMaapeptide insertionbinds CDato the
above proven AAV FOXPtherapeutic technologyPhase Igenerating a more effective AAVcvdLOX prFOXPvectoraka Kcardioproduct namefor transducing lymphocytes and macrophagesOur Kcardiowill also be tested in isolated human leukocytesT cells and Macfor improved gene delivery into these cellsgiving increased FOXPphenotypeegsecretion of ILand TGFbetamore Tregand for efficacy against
HCD induced atherosclerosis in LDLR KO miceThenwe will test this improved Kcardiovector in toxicology
studies in large animal modelsPhase IIAt the completion of this project we will be ready to launch our
AAVcvdLOX pr FOXPinto Phase I and II clinical trials in people with FH that are not controlled with
current therapy and predisposed to ASCVDMoreoverthere will be payoffs for this technology across medicineMostif not alldiseases of aging have inflammation as a major etiologiesarthritisAlzheimers and ParkinsonsetcThus the agents we develop here could have broad use across a wide range of diseasesnot only ASCVD Cardiovascular disease remains a major cause of morbidity and mortality for Americansparticularly for theof the population with familial hypercholesterolemiaFHwho have many fold higher coronary
mortalityEven in those with FHASCVD takes decades to developand adeno associated virusAAVbased
gene therapydocumented to last at leastyears in patientsis an ideal vector for treating preventing
ASCVDIn this fast track proposal we will generate a modified AAV vector carrying the powerful antiinflammatory humanh FOXPgenebut which only expresses hFOXPonly at the site of diseaseWe will
determine the level of AAV FOXPvirus need for efficacy against FH ASCVD in a mouse modelPhase Iand
then test for safetytoxicologyin large animals modelsCynomolgus monkeys and duroc pigletsOnce
passing the milestonesthe KcardioproductAAV LOX pr FOXPvector will be ready for Phase I and II
clinical trials