Safe, CRISPR/Cas-free B cell editing for therapeutic applications
Safe, CRISPR/Cas-free B cell editing for therapeutic applications
批准号:
10725412
负责人:
Michael R. Farzan
金额:
$22.13万
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-07-25 至 2025-06-30
关键词:
AddressAffinityAnimalsAntibodiesAntigensApoptosisAutomobile DrivingB-Cell Antigen ReceptorB-LymphocytesBiological AssayBiological ProductsCOVID-19 vaccineCell SurvivalCellsChromosome DeletionChromosome abnormalityClustered Regularly Interspaced Short Palindromic RepeatsConsumptionDNADNA cassetteDangerousnessDeletion MutagenesisDoseElementsEngraftmentEnzyme-Linked Immunosorbent AssayErythropoietinErythropoietin ReceptorEventExhibitsGene Transduction AgentGenesGeneticGenetic DiseasesGoalsHIVHIV envelope proteinHematocrit procedureHumanImmunoglobulin Class SwitchingImmunoglobulin Somatic HypermutationInfusion proceduresInsertional MutagenesisIntravenous infusion proceduresIntronsInverted Terminal RepeatLesionMeasuresMembraneMessenger RNAMethodsModelingMonitorMusNeoplastic Cell TransformationPathway interactionsPatientsPhysiologicalPrivatizationProliferatingProtocols documentationReagentRecombinant AntibodyRecombinant adeno-associated virus (rAAV)RecombinantsResearch PersonnelRetroviridaeRiskSafetySerotypingSerumSortingSystemTP53 geneTechniquesTerminal Repeat SequencesTestingTherapeuticTherapeutic antibodiesTranscriptTransgenesVaccinatedVaccinationadeno-associated viral vectorantibody testarmbase editingcellular engineeringcellular transductionchimeric antigen receptor T cellscostcost effectivedesignendonucleaseexomeexperimental studygene therapygenetic elementimprovedin vivoinfectious disease treatmentinterestneutralizing antibodynucleaserepairedresponseretroviral transductiontherapeutic genome editingtransgene expression
中文摘要
摘要
英文摘要
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.
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海外基金