mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables effificient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-ofexperiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specifific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentifified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profifile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarififies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNALNP-based antitumor immunotherapy. INTRODUCTION Messenger RNA (mRNA) vaccines are extremely versatile, as mRNA sequences can be easily tailored to encode any antigen of interest, enabling both rapid and mass-scale vaccine development against emerging pathogens as well as personalized vaccine design against cancers. Key to the success of the currently approved SARS-CoV2 mRNA vaccines and of many mRNA-based prophylactic vaccines in clinical development are lipid-based nanoparticles (LNPs), delivery vehicles that mediate effificient mRNA expression in situ and endow the vaccine with intrinsic adjuvant properties.  LNPs are composed of an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, with the ionizable lipid being considered the most important driver of mRNA expression. In contrast to their predecessors having a cationic charge across a wide variety of pH ranges, ionizable lipids are pH-sensitive lipids that endow the LNPs with a positive, membrane fusion-promoting charge at endosomal pH while having a neutral charge at physiological pH. The ionizable lipid is required to encapsulate the mRNA and drives endosomal escape. The PEGylated lipid improves LNP stability and controls the interaction of the LNPs with blood proteins and cells. Cholesterol and the phospholipid contribute to LNP stability and endosomal membrane destabilization. Intramuscular or subcutaneous administration of mRNA LNPs has been reported to elicit CD8 T cell responses against the mRNA encoded antigen. In t...

Few dispute that the rapid development and approval of two mRNA vaccines against COVID-19 was an unmitigated success, or that the evolving field of gene therapy has delivered multiple successful treatments for a range of rare diseases. Still, genomic medicines is a field that’s ripe for further innovation, in areas ranging from the development of improved delivery vehicles to the streamlining of manufacturing.   Three areas of genomic medicines could benefit greatly from an integrated approach to development: gene editing, gene therapy and mRNA medicines. All of these modalities hold great potential in the development of innovative therapies to treat cancer, autoimmune disease, rare inherited diseases and more. Engaging partners early in the development process is critical to assembling cutting-edge tools to enable efficient end-to-end development and manufacturing.   Customizing gene editing for improved efficiency CRISPR gene editing is being developed as a treatment for a range of applications, including CAR-T cells for cancer, gene-edited stem cells to treat diabetes and ex-vivo gene editing to treat the blood disorder beta thalassemia.   Integrated DNA Technologies (IDT)* can customize guide RNAs for common CRISPR research applications, as well as emerging gene-editing approaches like prime editing and research using the Cas13 enzyme. IDT also offers the Alt-R™ CRISPR-Cas9 System with the HiFi CRISPR-Cas9 nuclease, allowing for more precise gene editing with less of the off-target activity that can be a concern in gene editing.   Aldevron offers additional research-grade and GMP versions (SpyFiTM Cas9 Nuclease), which can help accelerate experimental therapies. Aldevron has partnered with external partners as well and can offer a full library of novel CRISPR nucleases to qualified customers, such as Eureca-V™ Nuclease, the wild-type MAD7™ CRISPR Type-V nuclease from Inscripta.   Improving the delivery of gene therapy Lipid nanoparticles (LNPs) are widely used to deliver mRNA, CRISPR components and other nucleic acids into cells, but developing the LNP chemistry and process is challenging. Precision NanoSystems (PNI) has advanced technologies that can ease the process of developing and manufacturing LNPs. Its GenVoy Delivery Platform includes a novel lipid library and LNP reagents optimized for key applications including vaccines, gene editing and cell therapies. And researchers can use PNI’s desktop NanoAssemblr® platform with NxGen™ microfluidics to make well-formed, stable LNPs using a production process that is scalable from small batch discovery research to GMP production.   The viral vectors used to deliver gene and cell therapies are also in high demand, intensifying the need for technologies that can ease development and manufacturing. Aldevron offers a range of plasmids essential for the development of viral vectors. And with the help of some key partnerships, Danaher’s life sciences compan...

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and rapid waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP (five (V) prolines, Flexibly-Linked, Inter-Protomer disulfide) was developed. Based on the original amino acid sequence of the wildtype spike, VFLIP was genetically engineered by using five proline substitutions, a flexible cleavage site amino acid linker, and an interprotomer disulfide bond. It has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X had detectable neutralizing antibody titers for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. The researchers's results indicate that the VFLIP-X delivered by circRNA induces humoral and cellular immune responses, as well as broad neutralizing activity against SARSCoV-2 variants. The study used the following products from SINOPEG: ionizable lipidoid SM-102 (Sinopeg), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Sinopeg), cholesterol (Sinopeg), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000, Sinopeg) A circular mRNA vaccine prototype producing VFLIP-X spike confers a broad neutralization of SARS-CoV-2 variants by mouse sera, Antiviral Research, Volume 204, 2022, 105370, ISSN 0166-3542, https://doi.org/10.1016/j.antiviral.2022.105370. (https://www.sciencedirect.com/science/article/pii/S0166354222001395)

L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion–enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. SINOPEG can supply linear PEG-maleimide: Chung S F ,  Kim C F ,  Tam S Y , et al. A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer[J]. Applied Microbiology and Biotechnology, 2020, 104(6). https://doi.org/10.1007/s00253-020-10484-4

Plant-derived cholesterol is the most important raw material component in mRNA vaccine production and gene therapy. As one of the key functional excipients of lipid nanoparticles (LNPs), cholesterol plays a role in mediating LNP endocytosis as well as stabilizing the LNP structure (cholesterol helps increase the fluidity or stiffness of cell membranes and the addition of cholesterol improves the stability of nanoparticles). Meanwhile, with the rapid development of related research, lipid-based drug delivery systems are becoming increasingly important in a wider range of therapeutic areas, including vaccines for infectious diseases, cancer immunotherapy, etc. SINOPEG supplies to the market plant-derived cholesterol products of non-animal origin without genetic risk, eliminating concerns about the risk of animal-derived cholesterol carrying animal viruses, and can be used in high-end formulation excipients: small molecule liposomal drugs, nucleic acid drugs, mRNA vaccines and non-animal-derived cell culture media for protein-based drugs.

Welcome to visit us at SINOPEG's Booth #2351 at BIO International Convention 2022, San Diego, USA from 13th to 16th June. SINOPEG is mainly engaged in drug delivery system (DDS) and new medical materials business. SINOPEG has a high-level team with strong technical reserve to research, develop and produce high-quality polyethylene glycol, polyethylene glycol derivatives, block copolymers, anti-diabetic drug side chains, lipid excipients (ingredients for mRNA delivery system), ADC and ProTAC Linker, etc. Please contact us at sales@sinopeg.com #BIO

Welcome to visit us at SINOPEG's Booth #137 at Tides USA 2022, Boston, USA from 9th to 12th May. SINOPEG has a high-level team with strong technical reserve to research, develop and produce high-quality polyethylene glycol, polyethylene glycol derivatives, block copolymers, anti-diabetic drug side chains, lipid excipients (ingredients for mRNA delivery system), ADC and ProTAC Linker, etc. Please coutact us at sales@sinopeg.com

Welcome to visit us at SINOPEG's Booth #137 at Tides USA 2022, Boston, USA from 9th to 12th May.