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Engineering Cell-Permeant Mini-Proteins With Enhanced Endosomal Escape for the Delivery of Active Therapeutics.
Engineering Cell-Permeant Mini-Proteins With Enhanced Endosomal Escape for the Delivery of Active Therapeutics.
상세정보
- 자료유형
- 학위논문(국외)
- 기본표목-개인명
- 표제와 책임표시사항
- Engineering Cell-Permeant Mini-Proteins With Enhanced Endosomal Escape for the Delivery of Active Therapeutics.
- 발행, 배포, 간사 사항
- 발행, 배포, 간사 사항
- 형태사항
- 125 p.
- 일반주기
- Source: Dissertations Abstracts International, Volume: 87-04, Section: B.
- 일반주기
- Advisor: Schepartz, Alanna.
- 학위논문주기
- Thesis (Ph.D.)--University of California, Berkeley, 2025.
- 요약 등 주기
- 요약The therapeutic potential of biologics remains largely constrained by the inability to efficiently deliver proteins into the cytosol, where many critical therapeutic targets reside. This dissertation addresses this fundamental challenge by engineering and characterizing cell-permeant mini-proteins (CPMPs) with enhanced endosomal escape properties, and by developing chemical tools to elucidate their intracellular trafficking mechanisms.In the first part of this work, a structure-guided approach was used to generate AV5.3, a rationally engineered derivative of the mini-protein ZF5.3. AV5.3 exhibits early endosomal escape through pH-triggered unfolding and achieves efficient cytosolic delivery of enzymatic cargos with reduced exposure to lysosomal degradation. Notably, AV5.3 successfully delivered active dihydrofolate reductase (DHFR), rescuing DHFR-deficient mammalian cells and demonstrating its potential as an enzyme replacement therapy.The second portion of this thesis demonstrates that ZF5.3 can be used to deliver RAS-binding monobodies into the cytosol of cancer cells, where they retain functional activity. This work expands the utility of CPMPs for targeting undruggable intracellular proteins and highlights their compatibility with chemically sensitive and functionally diverse cargo proteins.Finally, a novel photocatalytic proximity labeling platform was used to investigate the mechanisms of ZF5.3 uptake. By conjugating ZF5.3 to a blue-light-activated iridium photocatalyst, we aimed to map the proteins in close proximity to ZF5.3 during endocytic trafficking. While proteomic analysis confirmed engagement with clathrin-mediated pathways, limited reproducibility across replicates revealed the need for orthogonal methods and next-generation labeling platforms with improved specificity.Together, this work presents a comprehensive strategy for improving the cytosolic delivery of therapeutic proteins through protein engineering and mechanistic interrogation. These advances provide a strong foundation for developing next-generation delivery platforms and for targeting currently inaccessible intracellular disease pathways.
- 주제명부출표목-일반주제명
- 주제명부출표목-일반주제명
- 주제명부출표목-일반주제명
- 주제명부출표목-일반주제명
- 비통제 색인어
- 비통제 색인어
- 비통제 색인어
- 비통제 색인어
- 비통제 색인어
- 비통제 색인어
- 부출표목-단체명
- 기본자료저록
- Dissertations Abstracts International. 87-04B.
- 전자적 위치 및 접속
- 원문정보보기
MARC
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■020 ▼a9798293893423
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■040 ▼aMiAaPQ▼cMiAaPQ
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■1001 ▼aVazquez Maldonado, Angel Luis.
■24510▼aEngineering Cell-Permeant Mini-Proteins With Enhanced Endosomal Escape for the Delivery of Active Therapeutics.
■260 ▼a[S.l.]▼bUniversity of California, Berkeley. ▼c2025
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2025
■300 ▼a125 p.
■500 ▼aSource: Dissertations Abstracts International, Volume: 87-04, Section: B.
■500 ▼aAdvisor: Schepartz, Alanna.
■5021 ▼aThesis (Ph.D.)--University of California, Berkeley, 2025.
■520 ▼aThe therapeutic potential of biologics remains largely constrained by the inability to efficiently deliver proteins into the cytosol, where many critical therapeutic targets reside. This dissertation addresses this fundamental challenge by engineering and characterizing cell-permeant mini-proteins (CPMPs) with enhanced endosomal escape properties, and by developing chemical tools to elucidate their intracellular trafficking mechanisms.In the first part of this work, a structure-guided approach was used to generate AV5.3, a rationally engineered derivative of the mini-protein ZF5.3. AV5.3 exhibits early endosomal escape through pH-triggered unfolding and achieves efficient cytosolic delivery of enzymatic cargos with reduced exposure to lysosomal degradation. Notably, AV5.3 successfully delivered active dihydrofolate reductase (DHFR), rescuing DHFR-deficient mammalian cells and demonstrating its potential as an enzyme replacement therapy.The second portion of this thesis demonstrates that ZF5.3 can be used to deliver RAS-binding monobodies into the cytosol of cancer cells, where they retain functional activity. This work expands the utility of CPMPs for targeting undruggable intracellular proteins and highlights their compatibility with chemically sensitive and functionally diverse cargo proteins.Finally, a novel photocatalytic proximity labeling platform was used to investigate the mechanisms of ZF5.3 uptake. By conjugating ZF5.3 to a blue-light-activated iridium photocatalyst, we aimed to map the proteins in close proximity to ZF5.3 during endocytic trafficking. While proteomic analysis confirmed engagement with clathrin-mediated pathways, limited reproducibility across replicates revealed the need for orthogonal methods and next-generation labeling platforms with improved specificity.Together, this work presents a comprehensive strategy for improving the cytosolic delivery of therapeutic proteins through protein engineering and mechanistic interrogation. These advances provide a strong foundation for developing next-generation delivery platforms and for targeting currently inaccessible intracellular disease pathways.
■590 ▼aSchool code: 0028.
■650 4▼aChemistry.
■650 4▼aMolecular biology.
■650 4▼aCellular biology.
■650 4▼aBiochemistry.
■653 ▼aDelivery
■653 ▼aEscape
■653 ▼aMicroscopy
■653 ▼aPeptides
■653 ▼aProteins
■653 ▼aTherapeutics
■690 ▼a0485
■690 ▼a0307
■690 ▼a0379
■690 ▼a0487
■71020▼aUniversity of California, Berkeley▼bChemistry.
■7730 ▼tDissertations Abstracts International▼g87-04B.
■790 ▼a0028
■791 ▼aPh.D.
■792 ▼a2025
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17359340▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
