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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. Cationic HDL mimetics, termed nanolipoprotein particles (NLPs), can bind and complex with large replicon RNA molecules. Tuning the NLP composition and NLP-to-RNA ratios enabled control over formulation charge and stability. NLP-mediated in vivo delivery enhanced expression of encoded protein cargo and provides a readily functionalized, versatile platform for in vivo delivery of mRNA constructs. Unlabelled Image [ABSTRACT FROM AUTHOR]

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. Longterm 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 aMOMPprotein 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 nativeMOMPprotein. 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 membranebound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity. [ABSTRACT FROM AUTHOR]