Intracellular Trafficking of DNA for Gene Therapy
用于基因治疗的 DNA 细胞内运输
基本信息
- 批准号:10710840
- 负责人:
- 金额:$ 39.81万
- 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:2023-09-22 至 2027-08-31
- 项目状态:未结题
- 来源:
- 关键词:AffectAffinityAnimalsArchitectureBindingBinding SitesCREB1 geneCell NucleolusCell NucleusCell divisionCell membraneCellsComplexCytoplasmCytoskeletonDNADNA BindingDNA Binding DomainDNA Polymerase IDNA Polymerase IIDNA Polymerase IIIDNA SequenceDNA cassetteDNA deliveryDiseaseGene DeliveryGene ExpressionGene OrderGene TransferGenesGenetic MaterialsGenetic TranscriptionGoalsHost DefenseImmune responseImportinsIn VitroInflammationInflammatoryInflammatory ResponseInnate Immune SystemInterferonsInterphase CellLaboratoriesMediatingMessenger RNAMethodsMicrotubulesMotorMovementNF-kappa BNFIA geneNormal Statistical DistributionNuclearNuclear EnvelopeNuclear ImportNuclear Localization SignalPathway interactionsPatientsPlasmidsPolymeraseProductionProteinsRNA Polymerase IIRNA SplicingReceptor SignalingRibosomal RNASignal TransductionSiteStimulator of Interferon GenesTimeTissuesTransfectionTravelViralWorkcytokinedefense responseds-DNAeffective therapygene therapyimprovedin vivomechanical forceplasmid DNApromoterrelease factorresidencesensorsmall hairpin RNAtherapeutic genetraffickingtranscription factortransgene expression
项目摘要
Under almost all conditions using any method, the levels of gene transfer to any cell or tissue are low because
many barriers exist for the efficient delivery of genes to cells. The primary goal of our laboratory is to identify
and overcome the intracellular barriers to promote effective gene delivery and therapy. Exogenous viral or non-
viral DNA must cross the plasma membrane, travel through the cytoplasm and the cytoskeletal networks, cross
the nuclear envelope, localize to specific regions within the nucleus, and be transcribed in order for gene
therapy to be successful. We have shown that once in the cytoplasm, plasmids carrying DNA nuclear targeting
sequences (DTS) that are required for nuclear import in non-dividing cells rapidly associate with transcription
factors that mediate movement along microtubules and across the nuclear envelope. NF-kB is one such factor
that binds to several ubiquitously active DTSs and is required for DNA nuclear import, but in the cytoplasm it is
maintained in a sequestered state, unable to bind DNA. The question then is how is NF-kB activated to bind to
plasmids and mediate their cytoskeletal movement and nuclear import? In the case of NF-kB, a major pathway
for its activation is through a set of cytoplasmic dsDNA sensors, such as cGAS-STING, that are part of the
innate immune system and drive inflammatory responses. When dsDNA binds to cGAS, signaling cascades
are initiated that result in activation of key pro-inflammatory transcription factors (including NF-kB) and
ultimately production of pro-inflammatory cytokines. Thus, a major focus in the gene therapy space has been
to block activation of these sensors to reduce inflammation. However, we have observed that when cGAS is
silenced, cytoplasmically injected plasmids fail to traffic to the nucleus. We hypothesize that limited activation
of one or more of these sensors is actually needed for low level activation of key transcription factors in order
to facilitate DNA nuclear import in non-dividing cells. If we can find ways to limit sensor activation, but not
abolish it, this will allow for enhanced gene delivery with limited accompanying inflammation. We have also
spent considerable effort detailing the distribution of plasmids inside the nucleus and have found that the
subnuclear mislocalization of plasmids can affect their transcriptional activity. We have found that plasmids
localize to discrete transcriptional domains within the nucleus based on the type of promoter (Pol I, Pol II, or
Pol III) they carry and that when two different promoter types are placed on one plasmid, not only is the
intranuclear distribution of the DNA different that either promoter type alone, but transgene expression is
significantly reduced. We will dissect the pathways used for DNA movement within the nucleus and exploit
them to improve transgene expression based on the subnuclear localization of the transfected DNA. Our
specific aims are to (1) determine whether cytosolic dsDNA sensors are required for DNA nuclear import; (2)
evaluate whether residence time of DNA in the cytoplasm affects sensor activation and transfection efficiency;
and (3) characterize how subnuclear organization affects exogenous DNA expression.
在几乎所有条件下使用任何方法,基因转移到任何细胞或组织的水平都很低,因为
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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David A Dean其他文献
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{{ truncateString('David A Dean', 18)}}的其他基金
A multimodal delivery and treatment approach for Acute Lung Injury
急性肺损伤的多模式递送和治疗方法
- 批准号:
10378509 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
- 批准号:
10187645 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
- 批准号:
10631224 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
- 批准号:
10414888 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
Gene therapy for GERD-associated esophageal epithelial barrier dysfunction
GERD相关食管上皮屏障功能障碍的基因治疗
- 批准号:
10372106 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
A multimodal delivery and treatment approach for Acute Lung Injury
急性肺损伤的多模式递送和治疗方法
- 批准号:
10593959 - 财政年份:2020
- 资助金额:
$ 39.81万 - 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
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- 批准号:
10056811 - 财政年份:2020
- 资助金额:
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Novel Peptide/siRNA Nanoparticles for Treatment of Acute Lung Injury
用于治疗急性肺损伤的新型肽/siRNA纳米颗粒
- 批准号:
9376455 - 财政年份:2017
- 资助金额:
$ 39.81万 - 项目类别:
Development of a gene therapy approach to treat acute lung injury using a preclinical, large animal model
使用临床前大型动物模型开发治疗急性肺损伤的基因治疗方法
- 批准号:
9044084 - 财政年份:2016
- 资助金额:
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Cell-specific gene delivery methods for expression and silencing in the lung
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8978332 - 财政年份:2014
- 资助金额:
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