Document text
Principal Investigator: Michael R. Farzan
Organization: BOSTON CHILDREN'S HOSPITAL
Fiscal Year: 2024
Award: $265,500
Funding agency: National Institute of Allergy and Infectious Diseases
ABSTRACT
Both private entities and academic groups are pioneering B cell editing for therapeutic purposes,
either to express therapeutic antibodies from their native Ig loci, or other transgenes from an
ectopic locus. BCR editing is currently performed with CRISPR/Cas system and a homology-
directed repair template. Non-BCR trasngenes are introduced by retroviral transduction or
transposon-based random insertion. Both nuclease-based and insertion-based B cell engineering
techniques carry risks associated with chromosomal deletion (CRISPR/Cas) or insertional
mutagenesis (retroviruses/transposons). Furthermore, efficient editing is only possible by
isolating and editing B cells ex vivo meaning that therapies based on these editing protocols will
likely be very expensive.
We have discovered a method of B cell editing that requires no exogenous nucleases and does
not rely on random insertion. Our method relies on transducing class-switching B cells with a DNA
template supplied by a recombinant adeno-associated virus (rAAV) vector. The inverted terminal
repeat (ITR) sequences in the rAAV naturally integrate into double-strand breaks created by the
B cell class-switch machinery. With the right expression cassette designs, we can replace the
endogenous heavy chain variable (VH) segment or even express a non-antibody transgene from
within the BCR locus. This nuclease-free technique has potential advantages in terms of safety
and, because it requires only a single rAAV transduction event, it also promises a simple, cost
effective means of editing B cells in vivo.
Here we aim to develop our nuclease-free editing technique and provide proof-of-concept for
therapeutic applications. In Aim 1, we will optimize the design of our rAAV-delivered repair
template, and demonstrate the relative safety of our approach compared to CRISPR/Cas-based
editing. In Aim 2, we test different expression cassette designs for antibody and non-antibody
transgene expression and determine whether or not inclusion of cis-acting genetic elements that
increase somatic hypermutation can enhance affinity maturation of our edited B cells. In Aim 3,
we will address in vivo editing. We will determine whether or not in vivo nuclease-free editing
efficiency can be enhanced by vaccinating mice prior to rAAV administration to drive B cell class
switching and optimize our rAAV doses and timing relative to the pre-vaccination step. We will
also demonstrate the ability of our edited B cell system to produce recombinant antibodies (BCR
editing) and erythropoietin (non-antibody transgene expression) in mice.
Terms: <2019-nCoV vaccine><AAV vector><AAV-based vector><AIDS Virus><Aberrant Chromosome><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Address><Affinity><Animals><Antibodies><Antigens><Antioncogene Protein p53><Apoptosis><Apoptosis Pathway><Assay><Automobile Driving><B blood cells><B cell><B cell receptor><B cells><B-Cell Antigen Receptor><B-Cells><B-Lymphocytes><B-cell><Bioassay><Biological Agent><Biological Assay><Biological Products><Blood Serum><CAR T cells><CAR modified T cells><CAR-T><CAR-Ts><COVID-19 vaccine><CRISPR><CRISPR/Cas system><Cell Body><Cell Survival><Cell Viability><Cells><Cellular Tumor Antigen P53><Chromosomal Aberrations><Chromosomal Abnormalities><Chromosomal Alterations><Chromosomal Deletion><Chromosome Aberrations><Chromosome Alterations><Chromosome Anomalies><Chromosome Deletion><Chromosome abnormality><Class Switching><Class Switchings><Clustered Regularly Interspaced Short Palindromic Repeats><Consumption><Cytogenetic Aberrations><Cytogenetic Abnormalities><DNA><DNA Therapy><DNA cassette><Dangerousness><Deletion Mutagenesis><Deoxyribonucleic Acid><Dose><ECSF><ELISA><EPO-R><Elements><Engraftment><Enzyme-Linked Immunosorbent Assay><Epoetin><Erythropoietin><Erythropoietin Receptor><Event><Exhibits><Flanking Repeat Sequences><GUIDE-seq><Gene Therapy Vectors><Gene Transduction Agent><Gene Transduction Vectors><Gene Transfer Clinical><Genes><Genetic><Genetic Diseases><Genetic Intervention><Genome-wide Unbiased Identification of DSBs Enabled by sequencing><Goals><HIV><HIV envelope><HIV envelope protein><Hct><Hematocrit><Hematocrit procedure><Human><Human Immunodeficiency Viruses><IV Infusion><Ig Somatic Hypermutation><Immunoglobulin Class Switching><Immunoglobulin Class Switchings><Immunoglobulin Somatic Hypermutation><Infusion><Infusion procedures><Insertional Mutagenesis><Intervening Sequences><Intravenous infusion procedures><Introns><Inverted Terminal Repeat><Investigators><Isotype Switching><Isotype Switchings><LAV-HTLV-III><Lesion><Lymphadenopathy-Associated Virus><Measures><Membrane><Methods><Mice><Mice Mammals><Modeling><Modern Man><Monitor><Murine><Mus><Neoplastic Cell Transformation><Oncoprotein p53><P53><Packed Erythrocyte Volume><Packed Red-Cell Volume><Partial Monosomy><Pathway interactions><Patients><Phosphoprotein P53><Phosphoprotein pp53><Physiologic><Physiological><Privatization><Programmed Cell Death><Proliferating><Protein TP53><Protocol><Protocols documentation><Reagent><Recombinant Antibody><Recombinant adeno-associated virus><Recombinant adeno-associated virus (rAAV)><Recombinants><Research Personnel><Researchers><Retroviridae><Retroviruses><Risk><SARS-CoV-2 vaccine><SARS-coronavirus-2 vaccine><Safety><Serotyping><Serum><Severe Acute Respiratory Syndrome CoV 2 vaccine><Severe acute respiratory syndrome coronavirus 2 vaccine><Sorting><System><T cells for CAR><TP53><TP53 gene><TRP53><Techniques><Terminal Repeat><Terminal Repeat 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