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Customized and Modular Control of Gene Expression for Precision Gene Therapies


Mayfield, Acacia Michelle Hori (2024) Customized and Modular Control of Gene Expression for Precision Gene Therapies. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/d05v-0t68.


Genetic disorders are caused by mutations in essential genes that disturb the abundance or function of proteins, tipping cells and tissues from homeostatic harmony into disorder. Developing treatment for genetic diseases involves precision approaches, as gene therapies target the root causes of highly specific pathologic processes at the level of gene replacement, editing, or downstream compensation for a harmful genetic change. Safe access to these cell populations, and the ability to control the behavior of therapeutic cargo after delivery to target tissues, will enable the field to develop safe and effective therapies with the potential to be curative. Systemically delivered AAVs can noninvasively target therapeutic genetic cargo to diverse disease loci throughout the body, but at high doses required for therapeutic penetrance of naturally occurring serotypes, these vectors can cause severe toxicity, emphasizing the need for both targeted, efficient gene delivery vectors, and other means of transgene expression control. This work describes three examples of AAV capsid and cargo design strategies that seek to control where, when, and at what level therapeutic transgene expression can be achieved in a preclinical context. First, we utilize native putative regulatory elements to encourage physiologic level of ectopic frataxin expression in a mouse model of Friedreich’s Ataxia, finding that when delivered to both the brain and peripheral nervous system, treatment prevents progression of motor and coordination deficits. Next, we utilize the genetic incoherent feedforward loop circuit motif at the RNA level to decouple vector delivery level from transgene expression level of MeCP2 in a mouse model of Rett Syndrome, finding that when regulated to near endogenous healthy levels of RNA, AAV-MeCP2-IFFL enables behavioral rescue without overexpression toxicity. Lastly, we employ the mechanism for AAV-genome stability in vivo to modulate expression using a post-hoc AAV administration. Together, these methods and applications demonstrate that modular and custom approaches can improve the precision, safety and efficacy problems that the gene therapy field needs in order to advance more treatments for rare disorders.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Gene Therapy; Friedreich's Ataxia; Rett Syndrome
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Gradinaru, Viviana
Thesis Committee:
  • Kennedy, Mary B. (chair)
  • Elowitz, Michael B.
  • Lester, Henry A.
  • Gradinaru, Viviana
Defense Date:28 May 2024
Funding AgencyGrant Number
Chan Zuckerberg Initiative2018-191947
Aligning Science Across Parkinson's (ASAP)ASAP-020495
Merkin Translational Research Grant2021
NIH PioneerDP1NS111369A
Rett Syndrome Research TrustRSRT2024
Friedreich's Ataxia Research AllianceFARA.PHPS4FA
Record Number:CaltechTHESIS:05312024-185303498
Persistent URL:
Related URLs:
URLURL TypeDescription included in chapter 1 for chapter 3
doi: included in chapter 4
Mayfield, Acacia Michelle Hori0000-0001-7308-6480
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:16454
Deposited By: Acacia Mayfield
Deposited On:06 Jun 2024 23:26
Last Modified:08 Jul 2024 19:13

Thesis Files

[img] PDF (Redacted thesis (ch. 4 omitted)) - Final Version
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