# Biology of Subgenomic AAV Vector Particles

> **NIH NIH P01** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $464,029

## Abstract

PROJECT SUMMARY/ABSTRACT
Hemophilia A is an X-linked bleeding disorder caused by hereditary defects in the F8 gene (encoding coagulation
factor VIII, FVIII), which affects approximately 1 in 5000 male births worldwide. Clinically, hemophilia A is
manifested as a severe bleeding phenotype that typically requires costly protein replacement therapy. The recent
development of a gene therapy holds the promise of curing hemophilia A. Although the outcome of clinical trials
using AAV vectors for hemophilia A has been encouraging, Adeno-associated virus (AAV)-mediated delivery of
FVIII has not yet received regulatory approval. Major hurdles persist since there are concerns on gradually
decreasing levels of FVIII in circulation as well as clonal hepatocellular expansion in long-term studies in a canine
model, which raises concerns over genotoxicity or even cancer formation when using AAV vectors for human
gene therapy. Current AAV-FVIII gene delivery strategies (AAV-F8) follow a standard design, in which a mini-
promoter is used to drive F8 expression with two flanking ITRs. Nevertheless, clinical-grade vectors produced
were reasonably homogenous based on Southern blot analysis of vector genomes. Our new studies however,
using single molecule sequencing (SMRT), have uncovered a broad complexity of vector population that has
been missed using conventional methods of analysis. One notable subgenomic vector particle contains the
snapback genome (SBG), which may lead to dsRNA production in the host cells. For other SBGs having only
the promoter sequences, they run the risk of promoting oncogenic readthrough, which may be a substantial
safety concern. The central hypothesis of this project is that the combination of trans factors such as host cellular
proteins, viral helper functions and cis elements such as vector nucleotide compositions lead to SGP formation.
Therefore, a comprehensive mechanistic understanding of how these factors mediate SGP formation will be
essential for the development of new a vector production platform with reduced/eliminated SGP particles and for
the design of strategies to control and manage the potential hazards of SGP. To achieve these goals, we will
pursue the following aims: 1): To define the roles of trans factors in subgenomic particle formation; 2) to define
the role of vector genome composition in subgenomic particle formation; and 3) to develop strategies to
reduce/eliminate subgenomic particle formation.

## Key facts

- **NIH application ID:** 10778207
- **Project number:** 5P01HL160472-03
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** WEIDONG XIAO
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $464,029
- **Award type:** 5
- **Project period:** 2022-02-05 → 2027-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10778207

## Citation

> US National Institutes of Health, RePORTER application 10778207, Biology of Subgenomic AAV Vector Particles (5P01HL160472-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10778207. Licensed CC0.

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