Rendering Protein-Based Particles Transiently Insoluble for Therapeutic Applications

Herein we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery

Analysis of Venezuelan Equine Encephalitis Replicon Particles Packaged in Different Coats

Background: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. Methodology/Principal Findings: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. Conclusions/Significance: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules

GAG-specific ELISA analysis post boost.

<p>Sera was collected from individual animals 1 week after the booster vaccination and GAG-specific ELISA analysis was performed. Error bars represent 1 standard error.</p

An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer

Therapeutic anticancer vaccines are designed to boost patients’ immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression

GAG-specific ELISPOT analysis post boost.

<p>Splenocytes were isolated from individual animals and GAG specific gamma interferon ELISPOT assays were performed to determine the number of antigen-specific cytokine-secreting T cells. Error bars represent 1 standard error.</p

VEE GP coat mouse immunogenicity study design

a<p>Genome equivalent (GE) titer determined by quantitative reverse transcription PCR.</p>b<p>Infectious unit (IU) titer determined on Vero cells.</p

Structural protein coding region and location of glycoprotein mutations in different VEE viruses

<p>Structural protein coding region and location of glycoprotein mutations in different VEE viruses</p

Comparison of genome equivalents

a<p>Infectious unit (IU) titer determined on Vero cells. IU/ml titer represented.</p>b<p>Genome equivalent (GE) titer determined by quantitative reverse transcription PCR. GE/ml titer represented.</p