Host Cell Restriction Factors Blocking Efficient Vector Transduction: Challenges in Lentiviral and Adeno-Associated Vector Based Gene Therapies

**Background:** Gene therapy relies on efficient delivery of genetic material to patient cells, with lentiviral (LV) and adeno-associated virus (AAV) vectors being two of the most widely used systems. However, host cell restriction factors (RFs)—components of the innate and intrinsic antiviral immune systems—pose significant barriers to successful transduction. These RFs have evolved to protect organisms against infectious diseases and can recognize vector components at multiple stages, from cell entry to nuclear delivery. The review discusses how these RFs impact LV and AAV vector transduction and the implications for clinical gene therapy efficacy.

**Methods:** This is a narrative review synthesizing published literature on host RFs that block LV and AAV vector transduction. The review covers both pattern recognition receptors (PRRs) involved in innate immune sensing and intrinsic antiviral restriction factors. For LV vectors, the review draws extensively on knowledge from HIV-1 biology, as LV vectors are primarily derived from HIV-1. For AAV vectors, the review focuses on findings from gene therapy applications given AAV's non-pathogenic nature.

**Key Results:** For LV vectors, multiple RFs target different stages of transduction. RNA sensors include TLR7/8 (detecting ssRNA in endosomes), TLR3 (dsRNA), and RIG-I/MDA5 (cytoplasmic RNA sensors). DNA sensors include cGAS (recognizing stem-loop structures of ssDNA from HIV-1 cDNA), IFI16 (detecting incomplete HIV-1 DNA reverse transcripts), and DDX41 (binding DNA/RNA hybrids). Protein sensors include TLR2/4/10 recognizing viral glycoproteins. Intrinsic RFs include: TRIM5α (recognizes capsid, accelerates uncoating, prevents reverse transcription completion), APOBEC3G (cytidine deaminase causing G-to-A hypermutations during reverse transcription), MX2 (blocks nuclear translocation of viral DNA), SAMHD1 (dNTPase that depletes nucleotide pools for reverse transcription), and tetherin/BST-2 (entraps viral particles at the cell surface). SAMHD1 expression is high in hematopoietic stem/progenitor cells (HSPCs), explaining their resistance to LV transduction. TRIM5α expression levels are negatively correlated with LV vector transduction efficiency in human T-lymphocyte cell lines and CD34+ cells.

For AAV vectors, PRRs recognizing vector DNA include TLR9 (recognizes CpG DNA, signals through MyD88), cGAS (binds dsDNA >36 bp, activates STING-TBK1-IRF3 axis), IFI16 (binds ssDNA and dsDNA, can activate inflammasome), and AIM2 (binds dsDNA, forms NLRP3 inflammasome). RNA sensors RIG-I and MDA5 detect dsRNA intermediates generated from AAV ITR promoter activity. Capsid proteins are recognized by TLR2. Intrinsic RFs include: FKBP52 (when phosphorylated, binds the D-sequence within AAV ITR and inhibits second-strand DNA synthesis), PHF5A/U2 snRNP complex (recognizes incoming capsids and restricts after second-strand synthesis), and SUMOylation pathway proteins (Ubc9, Sae1, Sae2) that modify capsid protein VP2. Proteasomal degradation of AAV vectors is a major restriction mechanism, with EGF receptor tyrosine kinase phosphorylating AAV2 surface tyrosines leading to ubiquitination and degradation. Mutating key tyrosine residues on AAV2 dramatically increases transduction in vitro and in vivo. CRISPR screens identified GPR108 and TM9SF2 as critical host factors facilitating AAV transduction, and Crb3 as a key restriction factor in hepatocytes.

**Clinical Implications:** Host RFs represent major bottlenecks for both LV and AAV vector-based gene therapies. For LV vectors, high TRIM5α and SAMHD1 expression in HSPCs explains the difficulty in transducing these clinically important target cells. For AAV vectors, the prevalence of pre-existing immunity (35-80% of the human population depending on serotype) and the need for high vector doses exacerbate innate immune responses. Strategies to overcome these barriers include CpG depletion, incorporation of TLR- and cGAS-inhibitory sequences, expression of RIG-I-inhibitory proteins, use of immunosuppressants, and capsid engineering (e.g., mutating tyrosine residues to avoid phosphorylation and proteasomal degradation). The review emphasizes that data from wild-type viral infection cannot be directly translated to vector transduction, as vectors lack accessory/helper proteins that normally help evade RFs, but also are replication-incompetent, limiting the production of detectable viral components. Efforts to improve transduction efficiency toward the use of lower vector doses remain critical for improving clinical therapeutic efficacy.