Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors

Abstract

Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes.

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

“…Such a subunit is structurally homologous to the variable domains of the heavy chain-only antibodies (V HH ) found in camelids termed nanobodies. These small (of nanometric size) molecules were proven to have good stability, solubility, and tissue permeability which makes them superior reagents in various applications including cell biology, diagnostics, and therapeutic use [ 54 , 55 , 56 , 57 , 58 ]. We believe that the similar use of isolated variable subunits from common antibodies (V H or V L ), retaining high affinity to the antigen, in particular, in biochemical competition assays and imaging may result in higher mapping precision for ligand binding sites and image quality with faster sample processing.…”

Fantastic AAV Gene Therapy Vectors and How to Find Them—Random Diversification, Rational Design and Machine Learning

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Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

“…In a recent published work, nanobodies were used to retarget AAV2 to CD4 + cells. In total, five anti-human CD4 nanobodies were inserted into the hypervariable loop of the GH2/GH3 surface of VP2 or N -terminus of the VP1 capsid protein [ 134 ]. Arginines R585 and R588 were mutated to alanine to prevent binding to heparin sulphate proteoglycan.…”