Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression

Area of Science:

• Regenerative Medicine
• Gene Therapy
• Dermatology

Background:

• Inherited skin diseases like Netherton syndrome result from mutations in genes such as serine protease inhibitor Kazal-type 5 (SPINK5).
• Gene-modified skin grafts using lentiviral vectors offer potential therapeutic benefits for these conditions.

Purpose of the Study:

• To evaluate the long-term efficacy and stability of gene-modified skin grafts for Netherton syndrome.
• To investigate and overcome gene silencing phenomena observed with previous lentiviral vector constructs.

Main Methods:

• Lentiviral vector-mediated gene transfer of the SPINK5 gene into human keratinocyte stem cells.
• Generation of gene-modified human skin grafts and transplantation onto immunodeficient mice.
• Comparison of spleen focus-forming virus (SFFV) promoter-driven vectors with a minimal human involucrin promoter (INVOp) for SPINK5 expression.

Main Results:

• Initial vectors with a codon-optimized SPINK5 transgene and SFFV promoter led to robust skin reconstitution but experienced long-term gene silencing due to CpG methylation.
• Substitution with the INVOp, which has fewer CpG sites, prevented promoter silencing and resulted in durable, compartment-specific expression of lympho-epithelial Kazal-type-related inhibitor (LEKTI).
• Gene-modified skin grafts with the INVOp demonstrated sustained therapeutic protein production in vivo.

Conclusions:

• Lentiviral vectors utilizing the INVOp are a suitable platform for durable gene therapy of Netherton syndrome via gene-modified skin grafts.
• Codon optimization of transgenes can inadvertently introduce CpG sites, leading to promoter silencing, a critical consideration in vector design.
• This study highlights the importance of promoter selection to ensure long-term therapeutic gene expression in regenerative medicine applications.