Safe, CRISPR/Cas-free B cell editing for therapeutic applications
用于治疗应用的安全、无 CRISPR/Cas 的 B 细胞编辑
基本信息
- 批准号:10725412
- 负责人:
- 金额:$ 22.13万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-07-25 至 2025-06-30
- 项目状态:未结题
- 来源:
- 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
项目摘要
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.
抽象的
私人实体和学术团体都在开创用于治疗目的的 B 细胞编辑,
要么从其天然 Ig 位点表达治疗性抗体,要么从 Ig 基因座表达其他转基因
异位基因座。 BCR 编辑目前使用 CRISPR/Cas 系统和同源性进行
定向修复模板。非 BCR 转基因通过逆转录病毒转导或
基于转座子的随机插入。基于核酸酶和基于插入的 B 细胞工程
技术存在与染色体删除 (CRISPR/Cas) 或插入相关的风险
诱变(逆转录病毒/转座子)。此外,高效的编辑只能通过
离体分离和编辑 B 细胞意味着基于这些编辑方案的疗法将
可能非常昂贵。
我们发现了一种不需要外源核酸酶的 B 细胞编辑方法
不依赖于随机插入。我们的方法依赖于用 DNA 转导类别转换 B 细胞
模板由重组腺相关病毒(rAAV)载体提供。倒置端子
rAAV 中的重复 (ITR) 序列自然整合到由 rAAV 产生的双链断裂中。
B细胞类别转换机制。通过正确的表达盒设计,我们可以取代
内源重链可变 (VH) 片段或什至表达非抗体转基因
BCR 基因座内。这种无核酸酶技术在安全性方面具有潜在优势
而且,因为它只需要一个 rAAV 转导事件,所以它还承诺简单、成本低
体内编辑B细胞的有效手段。
在这里,我们的目标是开发我们的无核酸酶编辑技术并提供概念验证
治疗应用。在目标 1 中,我们将优化 rAAV 修复的设计
模板,并证明我们的方法与基于 CRISPR/Cas 的方法相比的相对安全性
编辑。在目标 2 中,我们测试了抗体和非抗体的不同表达盒设计
转基因表达并确定是否包含顺式作用遗传元件
增加体细胞超突变可以增强我们编辑的 B 细胞的亲和力成熟。在目标 3 中,
我们将讨论体内编辑。我们将确定是否进行体内无核酸酶编辑
在注射 rAAV 之前对小鼠进行疫苗接种可以提高效率,以驱动 B 细胞类别
相对于疫苗接种前步骤,切换并优化我们的 rAAV 剂量和时间。我们将
还证明了我们编辑的 B 细胞系统产生重组抗体 (BCR
编辑)和小鼠促红细胞生成素(非抗体转基因表达)。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Michael R. Farzan其他文献
Michael R. Farzan的其他文献
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