# Translational insights from nonclinical studies of AAV gene therapies for hemophilia: mechanisms underpinning variability and durability of gene expression

## TL;DR

This paper reviews how AAV gene therapy for hemophilia varies in effectiveness and longevity, focusing on factors like transcriptional efficiency and cellular stress.

## Contribution

The paper integrates preclinical and clinical data to identify mechanisms behind variability and durability in AAV gene therapy for hemophilia.

## Key findings

- Transcriptional efficiency, not vector genome copy number, is a key factor in treatment variability and durability.
- Factor IX gene therapy shows more stable long-term expression compared to factor VIII due to differences in cellular stress.
- Epigenetic modifications and protein-folding stress contribute to declines in transgene expression over time.

## Abstract

Adeno-associated virus (AAV) gene therapy is a promising approach for hemophilia, offering the potential for sustained therapeutic expression of coagulation factors. However, both variability and durability of transgene expression remain a challenge, limiting treatment predictability. Comparative preclinical and human liver biopsy studies suggest that transcriptional efficiency, rather than vector genome copy number (VCN), is a primary determinant of variability and durability in treatment response. Despite the presence of vector genomes in hepatocytes, transcriptional output varies significantly across species and individuals, indicating that VCN alone is insufficient to predict therapeutic efficacy. This review synthesizes findings from preclinical models (mice, dogs, non-human primates (NHPs), and human hepatocytes) and clinical liver biopsy studies to examine mechanisms influencing AAV gene therapy variability and durability. While vector genome retention is relatively comparable across species, transcriptional efficiency declines in higher species, particularly in NHPs, dogs, and humans. Beyond transcription, vector genome loss, hepatocyte turnover, immune responses, and cellular stress (e.g., endoplasmic reticulum (ER) stress) may contribute to intraindividual declines in transgene expression over time. Recent findings also highlight the role of epigenetic modifications, vector integration patterns, and translational shutdown linked to protein-folding stress in influencing durability. Expression patterns show greater long-term stability with factor IX (FIX) gene therapy compared to factor VIII (FVIII), which often declines more sharply. Distinctions may reflect differences in protein biosynthetic burden and cellular stress responses, particularly for FVIII. Most FIX trials use the highly active Padua variant, enabling lower expression levels with potentially less cellular stress, while the tendency of FVIII to misfold and trigger ER stress may contribute to transcriptional or translational shutdown over time. Integrating insights from preclinical models, human liver biopsies, and ongoing clinical trials, this review refines our understanding of AAV gene therapy variability and durability, ultimately guiding next-generation gene therapies to enhance long-term clinical efficacy.

Animal models of AAV gene therapy in hemophilia

Adeno-associated virus (AAV) based gene therapy is a promising new treatment for hemophilia. It works by using AAV as a delivery vehicle (vector) to carry the gene for the missing blood clotting protein, either factor VIII or factor IX, into the liver. Liver cells then use this genetic information to produce the clotting protein, which is released into the bloodstream. Gene therapy does not work the same for everyone. Some people make more clotting protein than others (variability), and in some, the effect lasts longer than in others (durability). Carefully conducted studies in animal models of hemophilia, as well as analyses of human liver biopsy samples after gene therapy, have helped us understand the reasons for these differences. Even when a similar amount of genetic material is delivered to the liver, the body’s ability to use it to make protein, a process involving transcription and translation, can vary. Several factors may influence how well and how long gene therapy works, including how the AAV vector enters the liver cell, the loss or turnover of liver cells, loss or suppression of AAV vectors over time, natural immune responses, and cellular stress. In this review, we summarize key insights gained from animal models, laboratory studies, and ongoing clinical trials to improve our understanding of how AAV gene therapy works for hemophilia.