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.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Digital-to-biological converter for on-demand production of biologics.

Manufacturing processes for biological molecules in the research laboratory have failed to keep pace with the rapid advances in automization and parellelization. We report the development of a digital-to-biological converter for fully automated, versatile and demand-based production of functional biologics starting from DNA sequence information. Specifically, DNA templates, RNA molecules, proteins and viral particles were produced in an automated fashion from digitally transmitted DNA sequences without human intervention.

Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity.

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.