Date of Award

2024

Embargo Period

7-31-2024

Document Type

Thesis

Degree Name

Master of Biomedical Science

Department

Molecular and Cellular Biology and Pathobiology

Additional Department

Pediatrics

College

College of Graduate Studies

First Advisor

Martin Kang

Second Advisor

Catrina Robinson

Third Advisor

Jeffery Jones

Fourth Advisor

Kris Helke

Fifth Advisor

John Baatz

Abstract

Therapeutic options for the treatment of Surfactant Protein-B (SP-B) deficiency, an autosomal recessive disorder that leads to fatal respiratory distress, are extremely limited with the exception of double lung transplantation. One method for treating this monogenic lung disease is with adeno-associated virus (AAV) gene therapy treatment that delivers the hSFTPB transgene into the lungs. The transient nature of AAV transgene expression led to a need for sustained gene expression, which could possibly be achieved through redosing. The main obstacle with redosing is the innate immune response and subsequent inflammatory response pathways that are activated after recognition of unmethylated CpG motifs in the AAV genome by toll-like receptor 9 (TLR9). Through readministration of an engineered AAV6.2FF vector with TLR9 inhibitory (TLR9i) sequences incorporated into the genome, prolonged survival in a mouse model of SP-B deficiency was demonstrated compared to a single dose of gene therapy. However, the specific gene or gene pathways impacted following TLR9 suppression have not been well characterized. I hypothesize that the incorporation of TLR9i sequences into the modified AAV6 vector genome will permit readministration through suppression of multiple immune response genes involved in the interferon pathways. This will be explored through two main aims: the production and purification of AAV vectors for assessment studies, and through exploration of gene expression analysis data to further characterize the immune response after repeat doses of the gene therapy treatment with and without TLR9 inhibition.

To assess the immune response following TLR9 inhibition, RNA was isolated from lung tissue in mice treated with single or repeat doses of AAV with (AAV-hSPBTLR9i) and without (AAV-hSPB) the TLR9i sequences, and analyzed by nCounter (Nanostring) gene expression analysis.

The gene expression analysis assessed over 700 different host response genes, with AAV-hSPB redosing treatment showing greater than 100 significant gene expression changes compared to less than 10 genes in mice redosed with AAV-hSPBTLR9i. Several genes were further examined including Serpina1a, Zbp1, Cxcl10, Stat1, and Mx1 due to their roles in regulating or responding to the immune response. Examination with qPCR, showed a significant increase in expression in genes involved in interferon response pathways including Mx1 and Stat1 following AAV-hSPB redosing but not AAV-hSPBTLR9i redosing. Another gene we examined was Tbx21 (T-bet), a marker for T helper cells (Th1) by immune cell profiling, which showed the highest levels after a repeat dose of AAV-hSPB but was reduced with AAV-hSPBTLR9i.

In conclusion, the AAV-hSPBTLR9i permits AAV redosing by suppressing multiple immune response genes including some involved in the interferon response pathway, as well as Th1 cells. For neonates with SP-B deficiency, the incorporation of the TLR9i sequences, and subsequent suppression of immune response genes, could allow for readministration of a gene therapy treatment to improve survival.

Rights

Copyright is held by the author. All rights reserved.

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