We have developed a new non-viral method for systemic delivery of antiviral siRNAs to T cells. Our results show that scFvCD7-9R is able to mediate efficient siRNA delivery to suppress HIV infection in both activated (Hu-PBL model) and naïve (Hu-HSC model) T cells. These findings overcome a critical barrier of in vivo delivery, significantly enhancing the prospect of siRNA-based therapeutics for HIV infection.
Since the first demonstration of in vivo gene silencing by hydrodynamic injection of siRNA (Song et al., 2003b
), there has been a concerted effort to develop more practical delivery strategies suitable for human therapy. A promising approach is to use targeting antibodies that undergo internalization after binding to surface receptors. To carry siRNA, antibodies can be coated on liposomes packaged with siRNA or fused to positively charged proteins/peptides that bind nucleic acids by charge interactions. Accordingly, an immunoliposome coated with antitransferrin scFv has been used to deliver HER-2 siRNA to tumor cells both in vitro and in vivo (Hogrefe et al., 2006
). Similarly, a HIV gp140 scFv fused to protamine could deliver siRNA to HIV-infected targets including primary T cells in vitro (Song et al., 2005
). A scFv-protamine fusion protein targeting the leukocyte-specific LFA-1 also delivered siRNA to primary human T cells in vitro (Peer et al., 2007
). In both studies, siRNA delivery to transplanted tumor cells expressing the targeted ligands was demonstrated in vivo, suggesting that the antibody-based approach may make in vivo siRNA delivery feasible. Our study confirms and extends these observations by using this strategy to deliver anti-viral siRNA in the context of an actual HIV infection. Our results show that siRNA binding capability can be conferred to scFvs by external disulphide conjugation to a 9R moiety. In addition to being relatively simple, this approach may also have an edge over recombinant fusion proteins, as expression of the positively charged residues might interfere with proper folding of the antibody during purification. Moreover, it allows use of the d-isoform of the peptide, which is relatively resistant to degradation by serum proteases (Hamamoto et al., 2002
). It is noteworthy that conjugation of the anti-CD7 antibody to 9R, a cell penetrating peptide (Kim et al., 2006
), did not affect its high level of T cell selectivity. Thus, it appears that after siRNA binding, the 9R component itself has no role in siRNA delivery, which is an advantage as non-specific transport into unintended cells is avoided.
Animal models for HIV-1 have suffered from either the lack of a system that precisely mirrors human HIV infection or, in the case of primate models, scarcity of the species, high cost, and the need to use the related but distinct simian virus for infection. We (L.S.) and others have recently developed gamma chain null mice that support long lasting HIV infection with both macrophage and T-cell tropic strains of HIV (Berges et al., 2008
; Watanabe et al., 2007
). Using the NOD/SCIDIL2γ−/−
mice, we found that HIV infection could be controlled both in a prophylactic setting, where viral challenge was performed after initiation of siRNA treatment, as well as in a post-infection therapeutic setting, where mice were reconstituted with PBLs from a human subject with an established HIV infection. Of note, knocking down CCR5 before viral challenge was not enough to completely block viral infection underscoring the importance of blocking multiple stages of viral replication by combinations of siRNAs targeting both host and viral genes. In a therapeutic setting, delivery to naïve/resting T cells will be important to ensure that siRNA is present in cells at the time of activation when they become most vulnerable to infection. This is also important for controlling viral resurgence in latently infected memory T cells. Thus, the successful delivery of siRNA to naïve T cells to control HIV infection in Hu-HSC mice attests to the versatility of our delivery strategy for clinical application.
It has been suggested that for a chronic infection like AIDS, a sustained antiviral state is best achieved by a gene therapy approach where vector-mediated delivery of shRNA to hematopoietic stem cells allows stable endogenous synthesis of siRNA in the repopulating progeny cells (Rossi et al., 2007
). HIV resistance has been demonstrated ex vivo in progeny T cells derived from shRNA transduced HSCs transplanted into SCID/Hu mice (Anderson et al., 2007
; Banerjea et al., 2003
; Brake et al., 2008
; Lee et al., 2005
; Rossi et al., 2007
; Scherer et al., 2007
; ter Brake et al., 2006
). However, obtaining stable transgene expression in sufficient numbers of expanded progeny which is critical for HIV-resistance has proved difficult to achieve in vivo (Levine et al., 2006
; Rossi et al., 2007
). A phase I clinical trial of a triple combination vector expressing an anti–tat/rev shRNA, a nucleolar localizing TAR decoy and an anti-CCR5 ribozyme has been launched recently which should shed light on the effectiveness of this approach and clarify concerns about toxicity related to shRNA expression, vector integration and the induction of interferon responses (Anderson et al., 2007
). Given the high mutability of HIV, another obvious disadvantage of vector-driven expression of a few specifically selected, but fixed shRNA sequences is that the protection would be compromised if escape mutants arise. In contrast, exogenous delivery of siRNA using the strategy described here not only delivers siRNA to a large proportion of T cells but also provides freedom to vary siRNA combinations to keep pace with the mutating virus if the need arises.
Nonspecific activation of the immune system and off-targeting effects have been reported with synthetic siRNA, however recent studies suggest that this can be overcome by optimizing the sequence or by chemical modifications (Svoboda, 2007
). Although over expression of shRNA in vivo has been reported to affect miRNA biogenesis and function leading to lethality in mice (Grimm et al., 2006
), a recent study suggests that repeated administration of synthetic siRNA targeting the liver did not affect liver-specific miRNA expression or function (John et al., 2007
). We also found that siRNA treatment did not affect the expression of several T cell expressed miRNAs. However, unlike for mouse liver-specific siRNAs, the gene targets for human T cell expressed miRNAs have not been definitively identified; hence we could only test one predicted target of miR-150 and found no changes in c-Myb protein levels after siRNA treatment. Thus, the risk of saturating endogenous miRNA pathway by exogenous siRNA appears to be minimal, although confirmation by a more comprehensive microarray/proteome analysis may be required. The possibility generating an immune response to the antibody used as the delivery vehicle is also a concern that needs to be addressed. However, many mAbs have been successfully used in clinical therapy without adverse effects and can also been ‘humanized’ in order to reduce potential toxicity (Marasco and Sui, 2007
). Further, since liposomal or polymeric nanoparticles can accommodate a lot more siRNA, use of siRNA encapsulated nanoparticles coated with CD7 scFv as a targeting agent could reduce the number of injections/dosage. While our own preparation did not induce TLR activation, the data needs to be reinforced by further testing in nonhuman primate models.
Another important issue in the treatment of HIV infection is the ability to target macrophages and dendritic cells. In this context, it has been recently reported that an antibody to LFA-1 may be able to target all leukocytes, although its potential for efficient siRNA delivery in vivo without adverse effects on leukocyte function remains to be tested (Peer et al., 2007
). Similarly, targeting approaches for siRNA delivery to other HIV-susceptible cell types could conceivably be used in combination with scFvCD7-9R. The availability of a preclinical animal model for HIV infection, as shown in this study, should allow rapid testing of these strategies, as well as other potential problems, such as viral escape and toxicity that have to be resolved before RNAi therapy can be translated for clinical use.