Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

Situ, Kathy; Chua, Bernadette A.; Bae, Song Yi; Meyer, Aaron S.; Morizono, Kouki

doi:10.1016/j.virol.2018.09.017

Cited by 7 publications

(6 citation statements)

References 69 publications

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“…Multiple strategies have been developed to render retargeted lentiviral vectors versatile by the addition of adapter molecules or antibodies. Sindbis pseudotyped LVs have been retargeted by the integration of biotin adaptor proteins, which bears the risk of unspecific adsorption to non-biotinylated proteins [ 34 ]. Moreover, avidin and streptavidin may bind to free biotin or cell-bound biotin, resulting in unspecific transduction.…”

Section: Discussionmentioning

confidence: 99%

“…Ad-LVs provide a higher level of selectivity compared to versatile targeting systems based on Sindbis pseudotyped LVs (on-to-off target ratios of 10–50-fold on cell lines and 25–115-fold on primary cells) [ 35 , 36 ]. In addition, retargeted GFP-encoding Sindbis-LVs transduced co-cultured cell lines above background levels even in the absence of adapters, while with Ad-LVs, the transduction efficiencies were below the detection limit [ 34 ]. Alphaviruses such as Sindbis-LVs enter cells via endocytosis, whereas paramyxoviruses fuse at the cell membrane, pointing towards the importance of alternative entry pathways for the selectivity.…”

Section: Discussionmentioning

confidence: 99%

“…The adapter size, avidity, and half-life can be adjusted to the targeted antigen and experimental needs. Additionally, natural ligands such as interleukins or growth factors may be considered as adapter molecules [ 34 , 39 ]. The Ad-LV technology may be employed by scientists in clinical and basic research to transduce cells lines or primary cells that are refractory to state-of-the-art LVs, e.g., VSV-G pseudotyped LVs.…”

Section: Discussionmentioning

confidence: 99%

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Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer

Cordes

Winter

Kolbe

et al. 2022

Viruses

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Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer

Cordes

Winter

Kolbe

et al. 2022

Viruses

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“…Chen and colleagues developed novel Sindbis E2 glycoprotein domains, responsible for receptor binding, by applying site-specific mutations to ablate native receptor tropism and direct addition of adapter binding molecules, such as the ZZ domain of protein A, avidin, biotin-adaptor peptide, and integrin-targeting peptide. [32][33][34][35][36][37][38][39] By mixing together LV and targeting ligand containing the adapter binding pair, such as Fc of antibody for ZZ domain, Sindbis-based LV could be redirected to a variety of receptors and cell types. A more recent extension of this strategy with Sindbis E2 was demonstrated using SpyTag and a disulfide bond-forming pair as adapter binding molecules for more permanent covalent incorporation of targeting ligand on viral surface.…”

Section: Introductionmentioning

confidence: 99%

Bispecific binder redirected lentiviral vector enables in vivo engineering of CAR-T cells

Huckaby

Landoni

Jacobs

et al. 2021

J Immunother Cancer

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BackgroundChimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity.MethodsHere, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3+ human T cells.ResultsThis redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor.ConclusionsThese findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy.

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“…This is because common LV vectors lack cell specificity: wild-type (WT) LV envelope proteins generally bind proteins ubiquitously present on the surface of most cells, leading to extensive off-target effects. Strategies to alter or restrict the natural tropism of LV vectors include either pseudotyping LV with different viral envelope proteins possessing altered tropism and biodistribution (3,12) or genetically inserting ligands, peptides, and single-chain antibodies (Abs) into viral envelope glycoprotein domains to confer new cellular specificity (13)(14)(15)(16)(17)(18)(19). Unfortunately, introducing large proteins can be deleterious to the structure of viral proteins, can impede proper folding of the incorporated peptide that diminishes cell binding, and may hinder viral infectivity by altering normal functions of viral attachment proteins or preventing conformational changes necessary for fusion (3).…”

mentioning

confidence: 99%

Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

Parker

Jacobs

Huckaby

et al. 2020

mBio

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Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

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Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

Cited by 7 publications

References 69 publications

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer

Bispecific binder redirected lentiviral vector enables in vivo engineering of CAR-T cells

Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

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