Biliang Hu

Redox-responsive nanocapsules for intracellular protein delivery

Direct delivery of proteins to the cytosol of cells holds tremendous potential in biological and medical applications. Engineering vehicles for escorting proteins to the cytosol in a controlled release fashion has thus generated considerable interest. We report here the preparation of redox-responsive single-protein nanocapsules for intracellular protein delivery. Through in situ interfacial polymerization, the target protein is noncovalently encapsulated into a positively-charged polymeric shell interconnected by disulfide-containing crosslinkers. The dissociation of the polymeric shell under reducing conditions and the subsequent release of protein were confirmed using cell-free assays in the presence of glutathione (GSH). The nanocapsules were demonstrated to be efficiently internalized into the cells and to release the protein in the reducing cytosol. Using the nanocapsule as a vehicle, we showed that active caspase 3 (CP-3) can be delivered and can induce apoptosis in a variety of human cancer cell lines, including HeLa, MCF-7 and U-87 MG. Our approach therefore presents an effective intracellular protein delivery strategy for therapeutic, diagnostic and reprogramming applications.

Protein nanocapsule weaved with enzymatically degradable polymeric network.

Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The protease-mediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.

HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells

Lentivectors (LVs) have attracted considerable interest for their potential as a vaccine delivery vehicle. In this study, we evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of human immunodeficiency virus (HIV) (LV-Gag) as a potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity is achieved through pseudotyping the vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the DC-SIGN protein. A single immunization by this vector induces a durable HIV Gag-specific immune response. We investigated the antigen-specific immunity and T-cell memory generated by a prime/boost vaccine regimen delivered by either successive LV-Gag injections or a DNA prime/LV-Gag boost protocol. We found that both prime/boost regimens significantly enhance cellular and humoral immune responses. Importantly, a heterologous DNA prime/LV-Gag boost regimen results in superior Gag-specific T-cell responses as compared with a DNA prime/adenovector boost immunization. It induces not only a higher magnitude response, as measured by Gag-specific tetramer analysis and intracellular IFN-γ staining, but also a better quality of response evidenced by a wider mix of cytokines produced by the Gag-specific CD8+ and CD4+ T cells. A boosting immunization with LV-Gag also generates T cells reactive to a broader range of Gag-derived epitopes. These results demonstrate that this DC-directed LV immunization is a potent modality for eliciting anti-HIV immune responses.

Immunization delivered by lentiviral vectors for cancer and infectious diseases.

Summary:  The increasing level of understanding of the lentivirus biology has been instrumental in shaping the design strategy of creating therapeutic lentiviral delivery vectors. As a result, lentiviral vectors have become one of the most powerful gene transfer vehicles. They are widely used for therapeutic purposes as well as in studies of basic biology, due to their unique characteristics. Lentiviral vectors have been successfully employed to mediate durable and efficient antigen expression and presentation in dendritic cells both in vitro and in vivo, leading to the activation of cellular immunity and humoral responses. This capability makes the lentiviral vector an ideal choice for immunizations that target a wide range of cancers and infectious diseases. Further advances into optimizing the vector system and understanding the relationship between the immune system and diseases pathogenesis will only augment the potential benefits and utility of lentiviral vaccines for human health.

Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity

We report a study of an integration-deficient lentiviral vector (IDLV) enveloped with a Sindbis virus glycoprotein mutant (SVGmu) capable of selectively binding to dendritic cells (DCs) for its potential as a vaccine carrier. The in vitro assays showed that the D64V point mutation in the catalytic domain of HIV-1 integrase efficiently inhibited the integration of the transgene upon vector transduction, while the targeting specificity of the vector to preferentially transduce and mediate durable expression in DCs was maintained. Substantial immune responses in C57BL/6 mice and complete protection against a challenge with the C57BL/6 thymoma EG.7 tumor expressing a delivered ovalbumin (OVA) antigen in mice have been achieved through the direct injection of the DC-directed IDLV encoding OVA. Thus, this DC-directed IDLV system represents a promising and efficient vector platform with remarkably improved safety for the future development of DC-based immunotherapy.

Serum Stable Natural Peptides Designed by mRNA Display

Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.

Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8(+) T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy.

Dendritic cell-directed lentivector vaccine induces antigen-specific immune responses against murine melanoma

Lentivectors are potential vaccine delivery vehicles because they can efficiently transduce a variety of non-dividing cells, including antigen-presenting cells, and do not cause expression of extra viral proteins. To improve safety while retaining efficiency, a dendritic cell (DC)-specific lentivector was constructed by pseudotyping the vector with an engineered viral glycoprotein derived from Sindbis virus. We assessed the level of anti-tumor immunity conferred by this engineered lentivector encoding the melanoma antigen gp100 in a mouse model. Footpad injection of the engineered lentivectors results in the best antigen-specific immune response as compared with subcutaneous and intraperitoneal injections. A single prime vaccination of the engineered lentivectors can elicit a high frequency (up to 10%) of gp100-specific CD8+ T cells in peripheral blood 3 weeks after the vaccination and this response will be maintained at around 5% for up to 8 weeks. We found that these engineered lentivectors elicited relatively low levels of anti-vector neutralizing antibody responses. Importantly, direct injection of this engineered lentivector inhibited the growth of aggressive B16 murine melanoma. These data suggest that DC-specific lentivectors can be a novel and alternative vaccine carrier with the potential to deliver effective anti-tumor immunity for cancer immunotherapy.

In Situ Modulation of Dendritic Cells by Injectable Thermosensitive Hydrogels for Cancer Vaccines in Mice

Attempts to develop cell-based cancer vaccines have shown limited efficacy, partly because transplanted dendritic cells (DCs) do not survive long enough to reach the lymph nodes. The development of biomaterials capable of modulating DCs in situ to enhance antigen uptake and presentation has emerged as a novel method toward developing more efficient cancer vaccines. Here, we propose a two-step hybrid strategy to produce a more robust cell-based cancer vaccine in situ. First, a significant number of DCs are recruited to an injectable thermosensitive mPEG–PLGA hydrogel through sustained release of chemoattractants, in particular, granulocyte-macrophage colony-stimulating factor (GM-CSF). Then, these resident DCs can be loaded with cancer antigens through the use of viral or nonviral vectors. We demonstrate that GM-CSF-releasing mPEG–PLGA hydrogels successfully recruit and house DCs and macrophages, allowing the subsequent introduction of antigens by vectors to activate the resident cells, thus, initiating antigen presentation and triggering immune response. Moreover, this two-step hybrid strategy generates a high level of tumor-specific immunity, as demonstrated in both prophylactic and therapeutic models of murine melanoma. This injectable thermosensitive hydrogel shows great promise as an adjuvant for cancer vaccines, potentially providing a new approach for cell therapies through in situ modulation of cells.

Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation

The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications.