Hepatic gene transfer for the treatment of hemophilia B and other genetic diseases

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Principal Investigator: Mark A Kay
Organization: STANFORD UNIVERSITY
Fiscal Year: 2024
Award: $701,277
Funding agency: National Heart Lung and Blood Institute

Adeno-associated viral vectors (rAAV) have shown promise in some liver-based clinical trials
including hemophilia B. However, one of the limitations is the loss of the vector genomes during
tissue growth and cell division limiting the duration of expression when treatment is initiated early
in life. For success in treating a number of genetic diseases treatment early in life is required. We
previously developed a non-nuclease mediated AAV-mediated homologous recombination (AAV-
HR) approach by using genomic homology arms to insert a protein coding sequence onto the end
of the endogenous Albumin gene such that after homologous recombination, the modified locus
would make a chimeric mRNA and both the endogenous albumin protein and a second protein,
in our case human factor IX and treated the bleeding diathesis in a murine model of hemophilia
B. Subsequently, this approach has been used to treat other murine models of human
hepatodeficiency disorders and a Phase I/II clinical trial for methylmalonic acidemia was initiated
by LogicBio therapeutics. The limitation is the efficiency of the process remains low and we have
recently established that off-target integration likely can produce a proportion of the therapeutic
protein. Our goal is to further study the genomic sources of the AAV-HR produced therapeutic
product as well as parameters that influence the efficiency of AAV-HR in cells and in vivo. To do
this, we will use high-throughput sequencing approaches to molecularly characterize the origin of
off-target transcribed RNAs produced after AAV-HR mediated transduction. We will establish if
they arise from vectors that integrate into regions of microhomologies or via non-homologous end
joining as well as identify which sets of these RNAs are translated. Our preliminary data suggests
that regions of the host genome that are more robust in R-loop formation will likely be more
efficient at AAV-HR. We will directly test this hypothesis and use the information to design
improved homology arms for AAV-HR. We will also do an unbiased in vivo genetic screen in mice
to establish which genes when expression is reduced results in higher AAV-HR. We will confirm
that parameters that influence non-nuclease mediated AAV-HR also effects nuclease mediated
AAV-HR. The results from these studies will not only provide new mechanistic insights into AAV-
HR but provide new approaches for enhanced genome editing, which would broaden the
application for treating human genetic diseases.

Terms: <21+ years old><AAV vector><AAV-based vector><Adult><Adult Human><Albumins><Amino Acids><Animals><Antibodies><Autoprothrombin II><Bleeding><Blood Coagulation Factor IX><Catalytic RNA><Cell Body><Cell Culture Techniques><Cell division><Cells><Christmas Disease><Christmas Factor><Clinical Trials><Coagulation Factor IX><Code><Coding System><Complementary DNA><Cultured Cells><DNA><Data><Deoxyribonucleic Acid><Diathesis><Disease><Disease susceptibility><Disorder><EC 3.4.21.22><Early treatment><Event><Exons><Factor IX><Factor IX Complex><Factor IX Deficiency><Factor IX Fraction><Factor VIII Deficiency><Gene Modified><Gene Targeting><Gene Transcription><Gene Transfer><Generalized Growth><Genes><Genetic Diseases><Genetic Screening><Genetic Transcription><Genome><Genomics><Goals><Growth><Hemophilia><Hemophilia A><Hemophilia B><Hemorrhage><Hepatic><Hepatic Cells><Hepatic Parenchymal Cell><Hepatocyte><Hereditary Disease><High-Throughput Nucleotide Sequencing><High-Throughput Sequencing><Human><Human Genetics><Inborn Genetic Diseases><Inherited disorder><Intervening Sequences><Introns><Knowledge><Left><Life><Liver><Liver Cells><Location><Maps><Mediating><Messenger RNA><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Murine><Mus><NHEJ><Non-Homologous End Joining><Non-Polyadenylated RNA><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Other Genetics><Patients><Phase 1/2 Clinical Trial><Phase I/II Clinical Trial><Plasma Thromboplastin Component><Process><Proteins><RIP seq><RIPseq><RNA><RNA Expression><RNA Gene Products><RNA immunoprecipitation and sequencing><RNA immunoprecipitation sequencing><Recombinant adeno-associated virus><Recombinant adeno-associated virus (rAAV)><Ribonucleic Acid><Ribosomes><Ribozymes><Scheme><Site><Source><Stop Codon><Technology><Termination Codon><Terminator Codon><Testing><Therapeutic><Tissue Growth><Transcript><Transcription><Transgenes><Translating><Translation Stop Signal><Work><adeno-associated viral vector><adeno-associated virus vector><adulthood><aminoacid><antihemophilic factor B><arm><blood loss><cDNA><cell culture><cell cultures><clinical applicability><clinical application><clinical relevance><clinically relevant><design><designing><early therapy><gene locus><gene modification><gene product><gene therapeutics><gene-based therapeutic><gene-based therapeutics><genes therapeutic><genes therapeutics><genetic condition><genetic disorder><genetic locus><genetically modified><genome editing><genomic editing><genomic location><genomic locus><hepatic body system><hepatic organ system><hepatoma cell><hereditary disorder><heritable disorder><homologous recombination><human model><humanized mice><humanized mouse><improved><in vivo><inborn error><inherited diseases><inherited genetic disease><inherited genetic disorder><insight><integration site><interest><liability to disease><mRNA><methylmalonic acidemia><methylmalonic aciduria><model of human><mouse model><murine model><nano pore><nanopore><native protein drug><neonatal mice><new approaches><novel approaches><novel strategies><novel strategy><nuclease><ontogeny><pharmaceutical protein><protein drug agent><protein-based drug><rAAV><recombinant AAV><sequencing platform><success><targeted sequencing><therapeutic gene><therapeutic protein><thromboplastinogen B><transgene><vector><vector genome>