Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms

Chimeric antigen receptor (CAR) T cells are a cancer immunotherapy of extremes. Unprecedentedly effective, but complex and costly to manufacture, they are not yet a therapeutic option for all who would benefit. This disparity has motivated worldwide efforts to simplify treatment. Among the proposed solutions, the generation of CAR T cells directly in the patient, i.e., in vivo, is arguably simultaneously the most technically challenging and clinically useful approach to convert CAR therapy from a cell-based autologous medicinal product into a universally applicable off-the-shelf treatment. Here, we review the current state of the art of in vivo CAR therapy, focusing especially on the vector technologies used. These cover lentiviral vectors and adenovirus-associated vectors as well as synthetic polymer nanocarriers and lipid nanoparticles. Proof of concept, i.e., the generation of CAR cells directly in mouse models, has been demonstrated for all vector platforms. Receptor targeting of vector particles is crucial, as it can prevent CAR gene delivery into off-target cells, thus reducing toxicities. We discuss the properties of the vector platforms, such as their immunogenicity, potency, and modes of CAR delivery (permanent versus transient). Finally, we outline the work required to advance in vivo CAR therapy from proof of concept to a robust, scalable technology for clinical testing. Chimeric antigen receptor (CAR) T cells are a cancer immunotherapy of extremes. Unprecedentedly effective, but complex and costly to manufacture, they are not yet a therapeutic option for all who would benefit. This disparity has motivated worldwide efforts to simplify treatment. Among the proposed solutions, the generation of CAR T cells directly in the patient, i.e., in vivo, is arguably simultaneously the most technically challenging and clinically useful approach to convert CAR therapy from a cell-based autologous medicinal product into a universally applicable off-the-shelf treatment. Here, we review the current state of the art of in vivo CAR therapy, focusing especially on the vector technologies used. These cover lentiviral vectors and adenovirus-associated vectors as well as synthetic polymer nanocarriers and lipid nanoparticles. Proof of concept, i.e., the generation of CAR cells directly in mouse models, has been demonstrated for all vector platforms. Receptor targeting of vector particles is crucial, as it can prevent CAR gene delivery into off-target cells, thus reducing toxicities. We discuss the properties of the vector platforms, such as their immunogenicity, potency, and modes of CAR delivery (permanent versus transient). Finally, we outline the work required to advance in vivo CAR therapy from proof of concept to a robust, scalable technology for clinical testing. Gene therapy is looking back at over 30 years of history and an ever-increasing number of therapeutic concepts and indications.1Dunbar C.E. High K.A. Joung J.K. Kohn D.B. Ozawa K. Sadelain M. Gene therapy comes of age.Science. 2018; 359eaan4672https://doi.org/10.1126/science.aan4672Crossref PubMed Scopus (569) Google Scholar Toward the end of the 2000s, initial success stories were soon followed by reports of severe side effects and deaths.2Somia N. Verma I.M. Gene therapy: trials and tribulations.Nat. Rev. Genet. 2000; 1: 91-99https://doi.org/10.1038/35038533Crossref PubMed Scopus (556) Google Scholar,3Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419https://doi.org/10.1126/science.1088547Crossref PubMed Scopus (2913) Google Scholar The field overcame these setbacks through technological innovation in vector design and has since then entered a booming phase with the present worldwide momentum involving academic laboratories, agile biotech companies, and big pharmaceutical players. The main driver for this development was an extension in indications from monogenetic diseases to complex acquired illnesses like cancer, which was facilitated by the use of genes encoding artificial proteins. The most prominent example is chimeric antigen receptors (CARs), which have revolutionized cancer immunotherapy with four medicinal products on the European market.4European Medicines AgencyKymriah: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/kymriah-epar-product-information_en.pdfDate: 2021Google Scholar, 5European Medicines AgencyTecartus: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/tecartus-epar-product-information_en.pdfDate: 2021Google Scholar, 6European Medicines AgencyYescarta: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/yescarta-epar-product-information_en.pdfDate: 2021Google Scholar, 7European Medicines AgencyAbecma: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/abecma-epar-product-information_en.pdfDate: 2021Google Scholar Still, major technological challenges for gene therapy remain: Besides the implementation of minimally invasive gene editing in treatment of monogenetic diseases and improving therapies’ safety profiles, cell-type-selective in vivo delivery of therapeutic genes is currently regarded as one of the problems with the highest priority.8Eisenstein M. Seven technologies to watch in 2022.Nature. 2022; 601: 658-661https://doi.org/10.1038/d41586-022-00163-xCrossref PubMed Scopus (1) Google Scholar The conversion of therapy-relevant cells into corrected or therapeutic protein-producing cells directly in the patient would make complex ex vivo manufacturing of genetically modified cells unnecessary and enable previously unviable therapeutic approaches. In vivo gene delivery has already reached the market. Based on adenovirus-associated vectors (AAVs) or oncolytic viruses, they rely on local application or the non-toxicity of their genetic cargo to offset the vectors’ broad cellular tropism. For many indications, however, the therapy-relevant cells form a small, precisely defined fraction, with off-target delivery to the majority of other cells being potentially deleterious. This is also true for CAR T cell therapy, which requires genetic modification of the patient’s T lymphocytes. CAR T cell therapy is a form of adoptive T cell therapy, in which cancer patients receive tumor-specific T cells that were genetically altered and expanded ex vivo. Chimeric antigen receptors (CARs) are composed of an extracellular antigen-binding domain, connected via a hinge region and a transmembrane domain to one or more intracellular signalling domains. Upon binding to their targets, CARs induce intracellular signaling that results in antigen-specific killing of the target cell and simultaneous proliferation of the CAR T cell.9June C.H. O’Connor R.S. Kawalekar O.U. Ghassemi S. Milone M.C. CAR T cell immunotherapy for human cancer.Science. 2018; 359: 1361-1365https://doi.org/10.1126/science.aar6711Crossref PubMed Scopus (1121) Google Scholar,10Sadelain M. CD19 CAR T cells.Cell. 2017; 171: 1471https://doi.org/10.1016/j.cell.2017.12.002Abstract Full Text PDF PubMed Scopus (57) Google Scholar Thus, CAR T cells can be regarded as living drugs that amplify in patients when they encounter target cancer cells. In recent years, this unique therapeutic concept has boosted research worldwide, with four products having been granted marketing authorization in Europe. Initial authorizations were granted to Yescarta and Kymriah, both targeting lymphoma cells via the B cell marker CD19. Besides lymphoma cells, healthy B lymphocytes are eliminated by these products, resulting in B cell depletion as a prominent side effect in treated patients.11Maude S.L. Laetsch T.W. Buechner J. Rives S. Boyer M. Bittencourt H. Bader P. Verneris M.R. Stefanski H.E. Myers G.D. et al.Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia.N. Engl. J. Med. 2018; 378: 439-448https://doi.org/10.1056/NEJMoa1709866Crossref PubMed Scopus (2162) Google Scholar Recently, two additional products, Tecartus and Abecma, have received regulatory approval, the latter extending indications for CAR therapy to multiple myeloma via B cell maturation antigen (BCMA).7European Medicines AgencyAbecma: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/abecma-epar-product-information_en.pdfDate: 2021Google Scholar The approval of the first CAR cell product not directed to CD19 marks an important step in this new field in cancer immunotherapy. Indeed, several hundred CAR T cell trials are ongoing worldwide, many of which aim at facilitating manufacturing and addressing additional malignancies.12Sterner R.C. Sterner R.M. CAR-T cell therapy: current limitations and potential strategies.Blood Cancer J. 2021; 11: 69https://doi.org/10.1038/s41408-021-00459-7Crossref PubMed Scopus (83) Google Scholar Use of CAR T cells in patients has started a public debate about cost explosions in health systems due to innovative therapeutics.13Gene therapies should be for all.Nat. Med. 2021; 27: 1311Crossref PubMed Scopus (3) Google Scholar CAR T cells are especially expensive, since they are individualized cell therapy products requiring time-consuming manufacturing procedures that rely on ex vivo gene transfer protocols. Following the isolation of lymphocytes from patients, the cells are activated and subsequently transduced, often using lentiviral or γ-retroviral vectors. The modified lymphocytes are then expanded and finally re-infused into the patient (Figure 1A ). This complex manufacturing process is expensive and, through the necessary manipulations of patients’ T lymphocytes, can alter their phenotype and activity.14Caruso H.G. Tanaka R. Liang J. Ling X. Sabbagh A. Henry V.K. Collier T.L. Heimberger A.B. Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function.J. Neurooncol. 2019; 145: 429-439https://doi.org/10.1007/s11060-019-03311-yCrossref PubMed Scopus (13) Google Scholar Moreover, T cells have to be isolated from the patient’s blood with utmost stringency, as the presence of residual tumor cells in the preparation during transduction may lead to accidental transfer of the CD19-CAR into leukemic cells during manufacturing. This resulted in a CAR T-cell-resistant clone, in which CD19 was masked by the single-chain variable antibody fragment (scFv) of the CAR, thus preventing its recognition. Cancer relapse and death of the patient followed.15Ruella M. Xu J. Barrett D.M. Fraietta J.A. Reich T.J. Ambrose D.E. Klichinsky M. Shestova O. Patel P.R. Kulikovskaya I. et al.Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.Nat. Med. 2018; 24: 1499-1503https://doi.org/10.1038/s41591-018-0201-9Crossref PubMed Scopus (260) Google Scholar Although only a single such case has been reported thus far, it highlights the complexities of the manufacturing process for CAR T cells that prevent their broad application in standard medical care as well as the risk associated with off-target transfer of CAR. Consequently, various paths are currently being pursued in preclinical and clinical research to improve CAR technology. Strategies aiming at facilitating the manufacturing process reach from automated systems16Lock D. Mockel-Tenbrinck N. Drechsel K. Barth C. Mauer D. Schaser T. Kolbe C. Al Rawashdeh W. Brauner J. Hardt O. et al.Automated manufacturing of potent CD20-directed chimeric antigen receptor T cells for clinical use.Hum. Gene Ther. 2017; 28: 914-925https://doi.org/10.1089/hum.2017.111Crossref PubMed Scopus (61) Google Scholar to allogeneic CAR T cells.17Qasim W. Allogeneic CAR T cell therapies for leukemia.Am. J. Hematol. 2019; 94: S50-S54https://doi.org/10.1002/ajh.25399Crossref PubMed Scopus (39) Google Scholar Although automation combined with the possibility to generate CAR T cells close to the patient’s bedside will greatly facilitate the logistics of manufacturing, this will not change the autologous, highly individualized nature of the product. In the allogeneic approach, CAR T cells prepared from a healthy donor are genetically manipulated to decrease their alloreactivity in the recipients. This is a step toward off-the-shelf CAR T cells, although the resulting products will most likely not be completely universal, owing to human leukocyte antigen barriers that necessitate adaptation to patient subgroups.18Smirnov S. Petukhov A. Levchuk K. Kulemzin S. Staliarova A. Lepik K. Shuvalov O. Zaritskey A. Daks A. Fedorova O. Strategies to circumvent the side-effects of immunotherapy using allogeneic CAR-T cells and boost its efficacy: results of recent clinical trials.Front. Immunol. 2021; 12: 780145https://doi.org/10.3389/fimmu.2021.780145Crossref PubMed Scopus (1) Google Scholar Graft-versus-host reactions as well as manufacturing complexity are circumvented when CAR T cells are generated directly in vivo. Here, a single, universally applicable medicinal product in the form of systemically administered vectors encoding the CAR would be used to transduce the patient’s T cells directly in their body. The resulting CAR T cells would be truly autologous (Figure 1). Although theoretically ideal for meeting the growing demand for CAR therapy, the in vivo approach has not yet made it to the clinic, mainly because suitable vector platforms were lacking, until recently. In the last five years, several groups have reported the successful in vivo generation of CAR T cells in mouse models (Table 1). Following advances in vector design, especially in vector targeting, these reports should be appreciated as breakthroughs in cancer immunotherapy and as showcases for the relevance of vector engineering in gene therapy in general.Table 1Preclinical data providing evidence for in vivo CAR T cell generationVectorMouse modelCARReferencePlatformTargeted receptorDosing and routebTU, transducing unit; i.p., intraperitoneal injection; i.v., intravenous injection; vg, vector genome.StraindNSG, NOD.Cg.PrkdcscidIL2rgtmWjl/SzJ; NCG, NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt; Albino B6, C57BL/6J-Tyrc−2J/J; B6, C57BL/6J.Immune transplantsePBMCs, human peripheral blood mononuclear cells; CB, cord blood-derived.Configurationfh, anti-human; m, anti-mouse.Target cellsgluc, luciferase expressing.LV with NiV glycoproteinsHuman CD82 × 106 TU (i.p.)NSGActivated human PBMCs (i.p.)hCD19-28ζ-CARRaji (i.p.)Pfeiffer et al.19Pfeiffer A. Thalheimer F.B. Hartmann S. Frank A.M. Bender R.R. Danisch S. Costa C. Wels W.S. Modlich U. Stripecke R. et al.In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome.EMBO Mol. Med. 2018; 10: e9158https://doi.org/10.15252/emmm.201809158Crossref PubMed Scopus (66) Google Scholar2 × 106 TU (i.v.)NSGHuman CD34+ CB cellshCD19-28ζ-CAREndogenous B cellsLV with NiV glycoproteinsHuman CD82.5 × 1011 particles (i.v.)NSGActivated human PBMCs (i.v.)hCD19-28ζ-CARNalm-6 luc, (i.v.)Agarwal et al.20Agarwal S. Weidner T. Thalheimer F.B. Buchholz C.J. In vivo generated human CAR T cells eradicate tumor cells.Oncoimmunology. 2019; 8e1671761https://doi.org/10.1080/2162402x.2019.1671761Crossref PubMed Google ScholarLV with MV glycoproteinsHuman CD44 × 1010 particles (i.p.)NSGActivated human PBMCs (i.p.)hCD19-28ζ-CAREndogenous B cellsAgarwal et al.21Agarwal S. Hanauer J.D. Frank A.M. Riechert V. Thalheimer F.B. Buchholz C.J. In vivo generation of CAR T cells selectively in human CD4+ lymphocytes.Mol. Ther. 2020; 28: 1783-1794https://doi.org/10.1016/j.ymthe.2020.05.005Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar1 × 1011 particles (i.v.)NSGActivated human PBMCs (i.v.)hCD19-28ζ-CARNalm-6 luc, (i.v.)4 × 1010 particles (i.v.)NSGHuman CD34+ CB cellshCD19-28ζ-CAREndogenous B cellsLV with NiV glycoproteinsHuman CD32 × 1011 particles (i.v.)NSGHuman CD34+ CB cellshCD19-28ζ-CAREndogenous B cellsFrank et al.22Frank A.M. Braun A.H. Scheib L. Agarwal S. Schneider I.C. Fusil F. Perian S. Sahin U. Thalheimer F.B. Verhoeyen E. Buchholz C.J. Combining T-cell-specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors.Blood Adv. 2020; 4: 5702-5715https://doi.org/10.1182/bloodadvances.2020002229Crossref PubMed Scopus (14) Google ScholarLV with SINV glycoproteinsHuman CD35 × 1010 particles (i.v.)NSGActivated human PBMCs (i.v.)hCD19-28ζ-CARBV-173 luc (i.v.)Huckaby et al.23Huckaby J.T. Landoni E. Jacobs T.M. Savoldo B. Dotti G. Lai S.K. Bispecific binder redirected lentiviral vector enables in vivo engineering of CAR-T cells.J. Immunother. Cancer. 2021; 9e002737https://doi.org/10.1136/jitc-2021-002737Crossref PubMed Scopus (5) Google ScholarAAV-DJN/AaN/A, not applicable.1 × 1011 vg (i.p.)NCGHuman PBMCs (i.p.)hCD4-28-4-1BBζ-CAREndogenous CD4 T cellsNawaz et al.24Nawaz W. Huang B. Xu S. Li Y. Zhu L. Yiqiao H. Wu Z. Wu X. AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia.Blood Cancer J. 2021; 11: 119https://doi.org/10.1038/s41408-021-00508-1Crossref PubMed Scopus (12) Google Scholar2 × 1011 vg (i.p.)NCGHuman PBMCs (i.p.)hCD4-28-4-1BBζ-CARMT-2 ATL (i.p.)NC with DNAMouse CD33 × 1011 particles/day on five consecutive days (i.v.cover 20 min with infusion pump.)Albino B6N/AmCD19-4-1BBζ-CARN/ASmith et al.25Smith T.T. Stephan S.B. Moffett H.F. McKnight L.E. Ji W. Reiman D. Bonagofski E. Wohlfahrt M.E. Pillai S.P.S. Stephan M.T. In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers.Nat. Nanotechnol. 2017; 12: 813-820https://doi.org/10.1038/nnano.2017.57Crossref PubMed Scopus (0) Google Scholar3 × 1011 particles/day on five consecutive days (i.v.cover 20 min with infusion pump.)Albino B6N/AmCD19-4-1BBζ-CAREμ-ALL(i.v.)NC with mRNAHuman CD86 weekly doses; 50 μg mRNA/dose (i.v.)NSGHuman T cells (i.v.)hCD19-28ζ-CARRaji-luc (i.v.)Parayath et al.26Parayath N.N. Stephan S.B. Koehne A.L. Nelson P.S. Stephan M.T. In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo.Nat. Commun. 2020; 11: 6080https://doi.org/10.1038/s41467-020-19486-2Crossref PubMed Scopus (48) Google Scholar6 weekly doses; 50 μg mRNA/dose (i.v.)NSGHuman T cells (i.v.)hROR1-4-1BBζ-CARLNCaP C42 prostate cancer cells (orthotopic)Human CD34 weekly bursts of 3 daily doses; 15 μg mRNA/dose (i.v.)B6N/AmCD19-28ζ-CAREμ-ALL (i.v.)LNP with mRNAMouse CD5Single dose of 10 μg LNPs (i.v.)B6N/AmFAP-28ζ-CAREndogenous fibrotic cardiac fibroblastRurik et al.27Rurik J.G. Tombácz I. Yadegari A. Méndez Fernández P.O. Shewale S.V. Li L. Kimura T. Soliman O.Y. Papp T.E. Tam Y.K. et al.CAR T cells produced in vivo to treat cardiac injury.Science. 2022; 375: 91-96https://doi.org/10.1126/science.abm0594Crossref PubMed Scopus (27) Google Scholara N/A, not applicable.b TU, transducing unit; i.p., intraperitoneal injection; i.v., intravenous injection; vg, vector genome.c over 20 min with infusion pump.d NSG, NOD.Cg.PrkdcscidIL2rgtmWjl/SzJ; NCG, NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt; Albino B6, C57BL/6J-Tyrc−2J/J; B6, C57BL/6J.e PBMCs, human peripheral blood mononuclear cells; CB, cord blood-derived.f h, anti-human; m, anti-mouse.g luc, luciferase expressing. Open table in a new tab Despite approved medicines and the breakthroughs in clinical research outlined above, gene therapies are far from being widely administered. Approved products address severe diseases where the risks associated with treatment are acceptable. As gene therapies begin to be considered for more indications, vectors need to be improved. This is especially relevant for systemic administration, which resulted in potentially dose-limiting toxicities, including but not limited to liver toxicities, as described for lipid nanoparticles (LNPs)28Hou X. Zaks T. Langer R. Dong Y. Lipid nanoparticles for mRNA delivery.Nat. Rev. Mater. 2021; 6: 1078-1094https://doi.org/10.1038/s41578-021-00358-0Crossref PubMed Scopus (167) Google Scholar, 29Sedic M. Senn J.J. Lynn A. Laska M. Smith M. Platz S.J. Bolen J. Hoge S. Bulychev A. Jacquinet E. et al.Safety evaluation of lipid nanoparticle-formulated modified mRNA in the sprague-dawley rat and cynomolgus monkey.Vet. Pathol. 2018; 55: 341-354https://doi.org/10.1177/0300985817738095Crossref PubMed Scopus (59) Google Scholar, 30Kedmi R. Ben-Arie N. Peer D. The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation.Biomaterials. 2010; 31: 6867-6875https://doi.org/10.1016/j.biomaterials.2010.05.027Crossref PubMed Scopus (264) Google Scholar, 31European Medicines AgencyOnpattro: EPAR - Product information.https://www.ema.europa.eu/en/documents/product-information/onpattro-epar-product-information_en.pdfDate: 2022Google Scholar and AAVs.32Food and Drug AdministrationBriefing Document Food and Drug Administration (FDA) Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) Meeting #70 Toxicity Risks of Adeno-Associated Virus (AAV) Vectors for Gene Therapy (GT) September 2-3, 2021.https://www.fda.gov/media/151599/downloadDate: 2021Google Scholar In a trial investigating treatment of X-linked myotubular myopathy with AAV8, all three patients receiving the higher dose experienced severe hepatotoxicity and two died.33Wilson J.M. Flotte T.R. Moving forward after two deaths in a gene therapy trial of myotubular myopathy.Hum. Gene Ther. 2020; 31: 695-696https://doi.org/10.1089/hum.2020.182Crossref PubMed Scopus (69) Google Scholar When organs other than liver are the intended target, high vector doses are required, since only a small fraction of the administered particles reaches the therapy-relevant cell type, as was impressively shown by human biodistribution data from two deceased children treated with Zolgensma, an AAV9-based gene therapy for the treatment of spinal muscular atrophy.34Thomsen G. Burghes A.H.M. Hsieh C. Do J. Chu B.T.T. Perry S. Barkho B. Kaufmann P. Sproule D.M. Feltner D.E. et al.Biodistribution of onasemnogene abeparvovec DNA, mRNA and SMN protein in human tissue.Nat. Med. 2021; 27: 1701-1711https://doi.org/10.1038/s41591-021-01483-7Crossref PubMed Scopus (8) Google Scholar Vectors that preferentially (or even exclusively) transduce the therapy-relevant cell type upon systemic administration are expected to reduce the required dose and toxicities.35Buchholz C.J. Friedel T. Büning H. Surface-engineered viral vectors for selective and cell type-specific gene delivery.Trends. Biotechnol. 2015; 33: 777-790https://doi.org/10.1016/j.tibtech.2015.09.008Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar For in vivo CAR therapy, use of such T-cell-targeted vectors is expected to prevent delivery of the CAR to non-T cells, especially cancer cells, avoiding fatal consequences for the patient. The degree of T cell-selectivity will have to be experimentally determined for any new type of T-cell-targeted vector. Receptor targeting is an approach of rational vector design that renders vectors selective for a defined cell surface receptor, e.g., for immune cell markers like CD3, CD4, CD5, or CD8. Importantly, selectivity is achieved at the level of cell entry. Accordingly, such vectors preferentially transduce cells displaying the targeted receptor on their surface. While some selectivity for organs of interest can be achieved by library-based empirical permutation of the chemical composition of nanoparticles36Dahlman J.E. Kauffman K.J. Xing Y. Shaw T.E. Mir F.F. Dlott C.C. Langer R. Anderson D.G. Wang E.T. Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics.Proc. Natl. Acad. Sci. U S A. 2017; 114: 2060-2065https://doi.org/10.1073/pnas.1620874114Crossref PubMed Scopus (89) Google Scholar, 37Paunovska K. Da Silva Sanchez A.J. Sago C.D. Gan Z. Lokugamage M.P. Islam F.Z. Kalathoor S. Krupczak B.R. Dahlman J.E. Oxidized cholesterol: nanoparticles containing oxidized cholesterol deliver mRNA to the liver microenvironment at clinically relevant doses.Adv. Mater. 2019; 311970098https://doi.org/10.1002/adma.201970098Crossref Google Scholar, 38Paunovska K. Gil C.J. Lokugamage M.P. Sago C.D. Sato M. Lando G.N. Gamboa Castro M. Bryksin A.V. Dahlman J.E. Analyzing 2000 in vivo drug delivery data points reveals cholesterol structure impacts nanoparticle delivery.ACS Nano. 2018; 12: 8341-8349https://doi.org/10.1021/acsnano.8b03640Crossref PubMed Scopus (47) Google Scholar, 39Liu S. Cheng Q. Wei T. Yu X. Johnson L.T. Farbiak L. Siegwart D.J. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.Nat. Mater. 2021; 20: 701-710https://doi.org/10.1038/s41563-020-00886-0Crossref PubMed Scopus (64) Google Scholar or residues in the AAV capsid,40Deverman B.E. Pravdo P.L. Simpson B.P. Kumar S.R. Chan K.Y. Banerjee A. Wu W.-L. Yang B. Huber N. Pasca S.P. Gradinaru V. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.Nat. Biotechnol. 2016; 34: 204-209https://doi.org/10.1038/nbt.3440Crossref PubMed Scopus (452) Google Scholar,41Byrne L.C. Day T.P. Visel M. Strazzeri J.A. Fortuny C. Dalkara D. Merigan W.H. Schaffer D.V. Flannery J.G. In vivo-directed evolution of adeno-associated virus in the primate retina.JCI Insight. 2020; 5e135112https://doi.org/10.1172/jci.insight.135112Crossref Google Scholar the incorporation of high-affinity binders, such as scFvs,42Zhou Q. Schneider I.C. Edes I. Honegger A. Bach P. Schönfeld K. Schambach A. Wels W.S. Kneissl S. Uckert W. Buchholz C.J. T-cell receptor gene transfer exclusively to human CD8(+) cells enhances tumor cell killing.Blood. 2012; 120: 4334-4342https://doi.org/10.1182/blood-2012-02-412973Crossref PubMed Scopus (37) Google Scholar or designed ankyrin repeat proteins43Frank A.M. Weidner T. Brynza J. Uckert W. Buchholz C.J. Hartmann J. CD8-Specific designed ankyrin repeat proteins improve selective gene delivery into human and primate T lymphocytes.Hum. Gene Ther. 2020; 31: 679-691https://doi.org/10.1089/hum.2019.248Crossref PubMed Scopus (7) Google Scholar (DARPins) into the particle structure in the course of receptor targeting has resulted in near-absolute cell type selectivity (>95%), especially for lentiviral vectors (LVs). LVs (Figure 2A ) are usually pseudotyped with the glycoprotein G of vesicular stomatitis virus (VSV-G), resulting in particles 120–150 nm in diameter. VSV-G mediates cell entry via the low-density lipoprotein receptor (LDLR) and its family members, which are expressed on many cell types.44Finkelshtein D. Werman A. Novick D. Barak S. Rubinstein M. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus.Proc. Natl. Acad. Sci. U S A. 2013; 110: 7306-7311https://doi.org/10.1073/pnas.1214441110Crossref PubMed Scopus (319) Google Scholar Accordingly, VSV-G pseudotyped LVs exhibit a broad tropism, achieving high transduction efficiencies on different human cell types, including activated T lymphocytes. CAR therapy mostly relies on lentiviral or γ-retroviral vectors for ex vivo transduction. In 2018, 54% of clinical studies in the US used LVs for CAR T cell generation.45Vormittag P. Gunn R. Ghorashian S. Veraitch F.S. A guide to manufacturing CAR T cell therapies.Curr. Opin. Biotechnol. 2018; 53: 164-181https://doi.org/10.1016/j.copbio.2018.01.025Crossref PubMed Scopus (133) Google Scholar Beyond that, over 100 registered clinical trials are ongoing, mostly using LVs in the context of immunological and hematological disorders and cancer.46Kohn L.A. Kohn D.B. Gene therapies for primary immune deficiencies.Front. Immunol. 2021; 12: 648951https://doi.org/10.3389/fimmu.2021.648951Crossref PubMed Scopus (11) Google Scholar,47clinicaltrials.gov. ClinicalTrials.gov, https://clinicaltrials.gov/, Last accessed: May 2022.Google Scholar Engineering of LV-incorporated envelope proteins for receptor targeting is a two-step approach consisting of (1) destroying natural receptor usage and (2) adding the binder for target recognition. Attempts to alter receptor usage of the VSV-G protein are ongoing48Anastasov N. Höfig I. Mall S. Krackhardt A.M. Thirion C. Optimized lentiviral transduction protocols by use of a poloxamer enhancer, spinoculation, and scFv-antibody fusions to VSV-G.Methods Mol. Biol. 2016; 1448: 49-61https://doi.org/10.1007/978-1-4939-3753-0_4Crossref PubMed Scopus (14) Google Scholar but challenging, since it mediates both receptor binding and membrane fusion. While the recent identification of the contact residues for LDLR has already facilitated the use of receptor-targeted VSV-LVs in the study of cellular interactions,49Nikolic J. Belot L. Raux H. Legrand P. Gaudin Y. A Albertini A. Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.Nat. Commun. 2018; 9: 1029https://doi.org/10.1038/s41467-018-03432-4Crossref PubMed Scopus (55) Google Scholar,50Yu B. Shi Q. Belk J.A. Yost K.E. Parker K.R. Huang H. Lingwood D. Davis M.M. Satpathy A.T. Chang H.Y. Systematic discovery of receptor-ligand biology by engineered cell entry and single-cell genomics.https://www.biorxiv.org/content/10.1101/2021.12.13.472464v1Date: 2021Google Scholar targeting approaches for LVs relying on glycoproteins from alpha- and paramyxoviruses, which have separate envelope proteins for binding and fusion, are more advanced (Figure 3A ).52Buchholz C.J. Mühlebach M.D. Cichutek K. Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer?.Trends. Biotechnol. 2009; 27: 259-265https://doi.org/10.1016/j.tibtech.2009.02.002Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Initially developed f