Identifying active and inhibitor-resistant MGMT variants for gene therapy

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA damage from alkylating agents like BCNU.
• MGMT is inhibited by O6-benzylguanine (O6BG), but the Pro140Lys (P140K) variant shows resistance.
• Current gene editing methods struggle to introduce the P140K variant into hematopoietic stem cells (HSCs).

Purpose of the Study:

• To identify novel MGMT variants resistant to O6BG and amenable to gene editing.
• To develop a screening platform for discovering functional DNA-repair protein variants.
• To enable in vivo enrichment of gene-modified HSCs for therapeutic strategies.

Main Methods:

• Computational analysis to select potential MGMT variants.
• Generation of a library of MGMT variant-expressing plasmids (pMGMTs).
• Functional screening in MGMT-deficient U251 cells using O6BG, pMGMT, and a reporter plasmid (mPlum_O6MeG), followed by flow cytometry.
• Validation using a second inhibitor, PaTrin-2.

Main Results:

• Identified active and O6BG-resistant MGMT variants through cell-based screening.
• Confirmed variant activity and inhibitor resistance using PaTrin-2.
• Found naturally occurring MGMT variants that are active and O6BG sensitive.
• Established a functional database of MGMT variants.

Conclusions:

• Developed a cell-based platform for screening DNA-repair proteins.
• Identified novel MGMT variants with potential for therapeutic applications in gene-edited HSCs.
• The findings pave the way for improved gene-editing strategies targeting DNA repair mechanisms.