We report a method of inducing antigen production in dendritic cells (DCs) by in vivo targeting with lentiviral vectors that specifically bind to the DC surface protein, DC-SIGN. To target the DCs, the lentivector was enveloped with a viral glycoprotein from Sindbis virus, engineered to be DC-SIGNspecific. In vitro, this lentivector specifically transduced DCs and induced DC maturation. A remarkable frequency (up to 12%) of ovalbumin (OVA)-specific CD8 + T cells and a significant antibody response were observed 2 weeks following injection of a targeted lentiviral vector encoding an OVA transgene into naïve mice. These mice were solidly protected against the growth of the OVA-expressing E.G7 tumor and this methodology could even induce regression of an established tumor. Thus, lentiviral vectors targeting DCs provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens.Immunization is one of the most productive tools in modern medical practice yet it still has limitations and novel methods of immunization are likely to be needed. 1 One of the great advances in our understanding of the process of immunization was the recognition of the role of dendritic cells (DCs) as specialized antigen-presenting cells (APCs). 2 This has led to attempts at DC-based immunization/vaccination involving loading DCs with specific antigens. 3, 4 Although significant progress has been made on various aspects of this vaccination method, many challenges still remain in order to rationally manipulate DCs to achieve protective immunity. [3][4][5] There is growing interest in genetically modifying DCs to make them either express antigens or produce immuno-stimulatory molecules. 6 Of these methods, viral vectors have been proven most effective for the delivery of genes into DCs in vitro. 7 The most popular viral vectors capable of transducing and expressing transgenes in DCs are adenoviral vectors, 8-10 gammaretroviral vectors 11, 12 and lentiviral vectors. [13][14][15] Considerable effort has also gone towards direct immunization using recombinant viral vectors as vaccine carriers. In these protocols, viral vectors are injected directly into an animal with the hope that a fraction will target immune cells and stimulate the desired immunity. Direct injection of adenoviral vectors was shown to induce both innate and adaptive immune responses and is currently being evaluated as a subunit vaccine for several infectious diseases and cancer. 16-18 Direct injection of lentivectors into mice does transduce DCs, leading to antigen-specific CD8 + T cell responses and anti-tumor immunity. 19-22 However, these 4Correspondence should be addressed to D.B. (baltimo@caltech.edu) or P.W. (pinwang@usc.edu). 3 These authors contribute equally to this work. COMPETING INTERESTS STATEMENT:The authors declare that they have no competing financial interests. Here we report a new method of in vivo DC targeting through the DC-specific surface molecule called DC-SIGN (also known as CD209) ...

Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient method to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and in vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral surface. This engineered vector could specifically bind to cells expressing surface immunoglobulin recognizing CD20 (␣CD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing ␣CD20. These results show the feasibility of engineering lentivectors to target immunoglobulin-specific cells to deliver a therapeutic effect. Such targeting lentivectors also could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology. 861

Purpose The purpose of this study was to investigate the potential of a T-cell-related targeting method using a lentiviral vector-based gene delivery system. Materials and Methods A lentiviral vector system was constructed by co-incorporating an anti-CD3 antibody (OKT3) and a fusogen into individual viral particles. The incorporation of OKT3 and fusogen was analyzed using confocal microscopy and the in vitro transduction efficiency was evaluated using flow cytometry. Blocking reagents (ammonium chloride (NH4Cl) and soluble OKT3 antibody) were added into vector supernatants during transduction to study the mechanism of this two-molecule targeting strategy. To demonstrate the ability of targeted transduction in vivo, Jurkat.CD3 cells were xenografted subcutaneously into the right flank of each mouse and the lentiviral vector was injected subcutaneously on both sides of each mouse 8 hours post-injection. Subsequently, the reporter gene (firefly luciferase) expression was monitored using a noninvasive bioluminescence imaging system. Results By co-displaying OKT3 and fusogen on the single lentiviral surface, we could achieve targeted delivery of genes to CD3-positive T-cells both in vitro and in vivo. Conclusions These results suggest the potential utility of this engineered lentiviral system as a new tool for cell type-directed gene delivery.

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody-antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand-receptor recognition mechanism for targeted modification of c-KIT receptor-expressing cells. For targeting, we incorporate membrane-bound human stem cell factor (hSCF), and for fusion, a Sindbis virusderived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in a SCF-receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell-specific surface receptors. Development of a high titer lentivector to receptor-specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non-target cells.

Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.