Cellular immune response to cryptic epitopes during therapeutic gene transfer

Area of Science:

• Immunology
• Gene Therapy
• Molecular Biology

Background:

• Immune responses often dictate gene therapy outcomes, typically triggered by transgenes or delivery vectors.
• Alternative reading frames (ARFs) within therapeutic cassettes are an underappreciated source of immune stimulation.

Purpose of the Study:

• To investigate if ARFs in a coagulation factor IX (F9) transgene can generate epitopes that elicit cytotoxic T lymphocyte (CTL) responses.
• To determine if these ARF-derived CTLs can eliminate F9-expressing hepatocytes in vivo.

Main Methods:

• Tested peptide candidates from an F9 ARF for CD8(+) T cell reactivity in human MHC-expressing mice.
• Administered adeno-associated virus (AAV) serotype 2 vectors with F9 transgene (AAV2/F9) to mice.
• Assessed CTL response against ARF epitopes and hepatocyte elimination in AAV2/F9 and codon-optimized AAV2/F9 (AAV2/F9-opt) treated mice.

Main Results:

• One peptide (p18) from the F9 ARF induced CD8(+) T cell reactivity.
• Systemic AAV2/F9 administration elicited a robust CTL response against the p18 epitope.
• ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but spared AAV2/F9-opt hepatocytes lacking the p18 epitope.

Conclusions:

• Cryptic epitopes generated from ARFs in therapeutic transgenes can unexpectedly elicit CTL responses.
• This mechanism poses a risk for adverse immune reactions in clinical gene therapy.
• Thorough sequence analysis for ARFs is crucial to mitigate immune-related adverse events in gene therapy protocols.