Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS

• Published in: Molecular therapy. Methods & clinical development Vol. 17; pp. 843 - 857
• Main Authors: Maturana, Carola J., Verpeut, Jessica L., Pisano, Thomas J., Dhanerawala, Zahra M., Esteves, Andrew, Enquist, Lynn W., Engel, Esteban A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.06.2020; American Society of Gene & Cell Therapy; Elsevier
• Subjects: AAV; Brain; CNS; Gene Delivery; Gene Promoter; Gene Therapy; iDisco; LAP; LAT; Latency-Associated Promoter; Latency-Associated Transcript; Light-Sheet Microscopy; Recombinant AAV; Retro-Orbital Injection; Spinal Cord; Viral vector; Brain; Viral vector; Gene Therapy; Gene Promoter; Latency-Associated Promoter; CNS; Recombinant AAV; Retro-Orbital Injection; iDisco; Latency-Associated Transcript; Spinal Cord; AAV; Gene Delivery; LAP; Light-Sheet Microscopy; LAT
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2020.04.004

Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration. [Display omitted] Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors. However, they have a limited payload capacity and lack small, long-lasting gene promoters for single-dose gene therapies. We identified small herpesvirus latency-associated promoters showing stable, pan-neuronal transgene transcription and translation in vivo for 6 months after AAV transduction.