Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8

Abstract

Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.

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“…[66][67][68] Knowledge from such studies can be utilized for the design of next-generation vectors equipped with receptor-specific linker molecules such as nanobodies or designed ankyrin repeat proteins (DARPins) which will increase the possibility for selective transduction. [69,70]…”

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“…In 2004, the Plückthun lab reported the first successful construction of DARPins designed to bind the maltose binding protein of Escherichia coli and eukaryotic protein kinases with high affinity and specificity [ 162 ]. Soon it was recognized that combining DARPins with viral vectors, e.g., lentivirus [ 30 , 163 ] or AAVs [ 30 , 138 , 139 , 164 , 165 ], could dramatically expand the range of applications, as this allows for the specific delivery of nucleic acids. Still, the challenges in translating such approaches to viral vectors are manifold, with the two largest hurdles perhaps being (i) the exposure of stably folded domains on the capsid surface that allows for a correct interaction with the receptor, and (ii) the interference of large insertions with viral capsid formation.…”

“…Finally, in addition to the successes with DARPin-armed AAVs in cancer research, two recent studies are noteworthy that aimed to further expand their applications to other targets. By inserting a murine CD8-specific DARPin into the GH2/3 loop of AAV2 VP1, an AAV-mCD8 vector was generated that targeted CD8 + cells in whole murine splenocytes with high efficiency (26-fold higher than unmodified AAV2) and >99% specificity [ 30 ]. Moreover, in a study by Hartmann and co-workers, interneurons were targeted by a GluA4-specific DARPin [ 165 ].…”

“…Non-covalent interactions can also be harnessed, for instance, by using an Fc-binding Z34C domain integrated into the AAV capsid (AAV-Z34C) [ 135 ] or a bispecific antibody that recognizes a conformational epitope [ 136 ] or a tag inserted into the AAV capsid (F(ab) 2 -AAV) [ 137 ]. Other molecules such as nanobodies (Nb) inserted into the GH2/GH3 loop of VP1 [ 29 ] or DARPins integrated into the same loop [ 30 ], fused to the VP2 N-terminus [ 138 , 139 ] or covalently linked [ 140 ] can also be used to efficiently retarget AAV vectors. This figure contains free clipart from (accessed on 1 April 2022).…”

“…Moreover, regardless of the technology used for cell and AAV isolation, the translatability of the isolated candidates to higher species is not always given. Another way to target specific cells is to make use of our knowledge about receptors and pathways to engineer ligands, antibodies, or small peptides that can be genetically or chemically coupled to the viral capsid [ 29 , 30 , 31 , 32 ]. While these rational approaches allow one to circumvent complicated and time-consuming high-throughput screens, their success is limited by the information on targeting molecules and target cells.…”