Preserving Genome Integrity In AAV-Mediated Gene Therapy
在 AAV 介导的基因治疗中保持基因组完整性
基本信息
- 批准号:10558679
- 负责人:
- 金额:$ 64.52万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2022
- 资助国家:美国
- 起止时间:2022-02-01 至 2026-01-31
- 项目状态:未结题
- 来源:
- 关键词:Animal ModelAnimalsAttentionBase SequenceBiologyBlood Coagulation DisordersCD8-Positive T-LymphocytesCanis familiarisCapsidCellsClonal ExpansionDNADNA IntegrationDNA RepairDNA Repair PathwayDNA StructureDataDependovirusDoseEffectivenessEventF8 geneFactor VIIIFatal OutcomeFrequenciesGene DeliveryGene ModifiedGene RearrangementGene therapy trialGenesGenetic DiseasesGenomeGenomicsGoalsHemophilia AHepatotoxicityHolliday Junction ResolvasesHumanImmune responseInsertional MutagenesisIntegration Host FactorsInverted Repeat SequencesInverted Terminal RepeatLinkLiteratureLiverLongitudinal StudiesMalignant NeoplasmsMediatingMethodsMicroRNAsModelingNatureNonhomologous DNA End JoiningOutcomeOutputPathway interactionsProductionProteinsReactionReportingRiskRisk ReductionSafetySamplingSequence AnalysisSmall Interfering RNAStructureTherapeuticTimeTissuesToxic effectTransgenesTreatment EfficacyValidationViralViral GenomeWorkadeno-associated viral vectorcell growthclinical developmentdeep sequencingdesigndog genomeexperiencegene correctiongene productgene therapygenome integritygenotoxicityhomologous recombinationimprovednext generation sequencingnovelnovel strategiesnucleaseparticlepreservationpreventrepairedsmall hairpin RNAsmall moleculesmall molecule inhibitortherapeutic genetransduction efficiencytransgene expressionvectorvector genomeviral DNA
项目摘要
ABSTRACT
Adeno-associated virus (AAV) vectors are in clinical development for delivery of genes to treat multiple genetic
diseases including hemophilia. While progress has been made to optimize gene delivery, in some studies the
required AAV vector doses were high, leading to toxicity and even fatal outcomes in one study. These findings
highlight the need for novel approaches to reduce the AAV vector dose to minimize liver toxicity, anti-AAV
immune responses, and genotoxicity. Our recent studies and work from others have identified an
underappreciated limitation to efficient gene correction with AAV vectors. In a long term study of AAV gene
delivery of FVIII in hemophilia A dogs, we found that most of the AAV vector genomes were highly rearranged
in transduced liver tissues. These rearrangements typically disrupted the transgene, and so would compromise
expression of the transgene product—unexpectedly, our data indicated that most of the AAV vector genomes
present did not produce functional protein after transduction. These rearranged AAV genomes were present in
integrated forms but also in AAV concatemers that may be episomal forms. It is unclear whether these
rearrangements occurred during vector production or after transduction of the target cells, though data is
accumulating that at least some of the rearrangements originate in vector producer cells. Our hemophilia A dog
study also identified integration events in the canine genome within genes linked to cell growth and cancer that
were associated with clonal expansions. Validation of integrated AAV DNA in these expanded clones by
sequence analysis showed that in all cases integrated vectors were highly rearranged, with only one of five
encoding an intact transgene. An extensive literature documents interactions of AAV with host DNA repair
pathways in both vector producer and target cells, though the influence of host factors in AAV DNA
rearrangements is mostly unstudied. We hypothesize that modulation of host cell pathways can suppress AAV
DNA rearrangements, thereby allowing improved transgene expression per vector DNA copy. In this proposal,
we will 1) implement a deep sequencing method to quantify rearrangement frequency in a statistically rigorous
fashion, 2) identify cellular pathways that can be modulated with small molecules, siRNAs, or microRNAs that
suppress vector rearrangements, and 3) devise novel delivery strategies that support efficient pathway
modulation, suppress vector rearrangement, and boost transgene output per vector copy. These methods will
be assessed during AAV vector production (Specific Aim 1) and after AAV delivery in the transduced target cells
(Specific Aim 2). Our deliverables at the end of the project will be a greatly enhanced understanding of the
interaction of AAV with host cell DNA handling pathways, and methods for modulating these pathways to allow
safe and effective gene delivery at lower vector doses.
抽象的
腺相关病毒 (AAV) 载体正处于临床开发阶段,用于传递基因来治疗多种遗传性疾病
虽然在优化基因传递方面取得了进展,但在一些研究中
在一项研究中,所需的 AAV 载体剂量很高,导致毒性甚至致命。
强调需要新的方法来减少 AAV 载体剂量,以尽量减少肝毒性、抗 AAV
我们最近的研究和其他人的工作已经确定了一个。
在 AAV 基因的长期研究中,AAV 载体有效基因校正的局限性未被充分认识。
在A型血友病狗体内传递FVIII时,我们发现大多数AAV载体基因组发生了重新排列
在转导的肝组织中,这些重排通常会破坏转基因,因此会受到损害。
转基因产物的表达——出乎意料的是,我们的数据表明大多数 AAV 载体基因组
这些重排的 AAV 基因组在转导后不产生功能性蛋白质。
整合形式,但也存在可能是游离形式的 AAV 串联体中,目前尚不清楚这些是否存在。
重排发生在载体生产期间或靶细胞转导之后,尽管数据是
累积至少一些重排起源于我们的血友病A狗。
研究还发现了犬基因组中与细胞生长和癌症相关的基因中的整合事件
与这些扩增克隆中整合的 AAV DNA 的验证有关。
序列分析表明,在所有情况下,整合载体都高度重新排列,只有五分之一
编码完整转基因的大量文献记录了 AAV 与宿主 DNA 修复的相互作用。
尽管 AAV DNA 中宿主因素的影响,载体生产者和靶细胞中的途径
我们大胆地认为,调节宿主细胞通路可以抑制 AAV。
DNA 重排,从而改善每个载体 DNA 拷贝的转基因表达。
我们将1)实施深度测序方法,以技术严格的方式量化重排频率
时尚,2) 识别可以用小分子、siRNA 或 microRNA 调节的细胞通路,
抑制载体重排,3) 设计支持有效途径的新颖递送策略
这些方法将调节、抑制载体重排并提高每个载体拷贝的转基因输出。
在 AAV 载体生产期间(具体目标 1)以及 AAV 递送至转导的靶细胞后进行评估
(具体目标 2)。项目结束时我们的交付成果将大大增强对项目的理解。
AAV 与宿主细胞 DNA 处理途径的相互作用,以及调节这些途径以允许
以较低的载体剂量安全有效的基因传递。
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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Frederic D Bushman其他文献
Frederic D Bushman的其他文献
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{{ truncateString('Frederic D Bushman', 18)}}的其他基金
mVACS--mRNA Vaccines for C. difficile Suppression
mVACS--用于抑制艰难梭菌的 mRNA 疫苗
- 批准号:
10625573 - 财政年份:2023
- 资助金额:
$ 64.52万 - 项目类别:
Preserving Genome Integrity In AAV-Mediated Gene Therapy
在 AAV 介导的基因治疗中保持基因组完整性
- 批准号:
10338480 - 财政年份:2022
- 资助金额:
$ 64.52万 - 项目类别:
Linking insertional mutagenesis and cell function to improve CAR T cell therapy
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- 批准号:
10398224 - 财政年份:2019
- 资助金额:
$ 64.52万 - 项目类别:
Linking insertional mutagenesis and cell function to improve CAR T cell therapy
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