Medicines that are based on mRNAs and proteins – particularly vaccines and antibodies – are time-intensive and costly to make. The challenge is in producing the necessary mRNAs and proteins in large enough amounts at low cost.

Now, researchers at the Lewis Katz School of Medicine at Temple University have discovered a new way to boost protein production in cells by as much as several hundredfold. The findings, published online in the journal Molecular Therapy, could lead to the generation of a universal booster for protein production for the development of bioproducts, drugs, and vaccines.

“We are very excited about our discovery, given its potential to increase protein production for a broad range of applications across many fields and areas of research,” explained Wenhui Hu, MD, PhD, Professor of Neuroscience and Pathology in the Center for Metabolic Disease Research and the Department of Pathology at the Lewis Katz School of Medicine. “It should be very useful for the production of therapeutic proteins like antibodies and mRNA vaccines.”

The new breakthrough centers on Exin21, a sequence of 21 nucleotides that encodes a short peptide, designated Qα. Exin21/Qα, when added to the coding region of a targeted gene, possesses a unique ability to greatly increase the production of a specific protein molecule. The mechanism behind this increase lies in the promotion of mRNA synthesis and stability and protein expression and secretion.

Dr. Hu and colleagues made the groundbreaking discovery during an exploratory study related to COVID-19 and its causative virus, SARS-CoV-2. “At the time, we were trying to identify ways to enhance the expression of SARS-CoV-2 viral proteins in mammalian cells,” Dr. Hu said. “During the process of molecular cloning, we accidentally found Exin21, which could boost the production of not only multiple SARS-CoV-2 viral proteins but also many other proteins.”

In addition to showing that Exin21/Qα can increase the production of SARS-CoV-2 antibody, the researchers demonstrated that adding Exin21/Qα to any mRNA can dramatically increase the production of the corresponding protein. This was the case for many different types of proteins, including viral, nonviral, intracellular, structural, and secretory proteins.

Exin21/Qα was also found to increase the efficiency of viral packaging, the process by which viruses assemble the protein shell that stores their genetic material. Any inefficiencies in this process can have a significant impact on research involving viruses and pseudoviruses, particularly in the context of studying antibodies and vaccines against SARS-CoV-2 variants or emerging viruses. Exin21/Qα effectively addresses this bottleneck, making it an asset for viral studies.

“Our findings with Exin21/Qα are applicable to so many proteins that our research could lead to a paradigm shift in the production of mRNA and protein-based therapeutics,” Dr. Hu said. “Being only 21 base pairs in length makes Exin21 universally accessible and useful for researchers in academia and industry.”

The research team is now working to further optimize the use of Exin21 and similar motifs for boosting different types of antibodies and mRNA vaccines. They aim to extend their research beyond cell models to gain a deeper understanding of its function in living systems.

“Although the comprehensive underlying mechanisms remain to be elucidated, perhaps, similar coding-motifs, such as Exin24 or Exin27, could be identified in the future,” Dr. Hu added.

 

Chinese News

2023-03-02 12:21

上海

来源：澎湃新闻·澎湃号·湃客

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为mRNA及蛋白疗法扫除障碍：胡文辉团队开发稳定mRNA并促进其表达的新技术

How do you study something complex like HIV-associated neurocognitive disorders (HAND) when you can’t collect the one thing that is critical to the research – live brain tissue?

If you’re a scientist, you create a model.

Recently, two researchers from the Lewis Katz School of Medicine received a $3.4 million grant from the National Institutes of Health to generate human brain organoids, or “mini-brains,” on which they will study the impact of HIV infection and methamphetamine (meth) on the central nervous system.

Mini-brains are 3D tissue structures generated from pluripotent stem cells in a test tube. The mini-brains contain different cell types and represent the anatomical structure of an actual brain.

“These mini-brains provide physiologically relevant tissue for the study of HAND, something which has been hampered in the past by difficulties in collecting and culturing live brain tissues,” says Wenzhe Ho, MD, MPH, a Professor in the Department of Pathology and Laboratory Medicine and in the Center for Substance Abuse at Temple.

“Unlike mini-brain models developed by other investigators, we will generate one that not only contains the ordinary brain cells, but also the resident immune cells – called microglia – which are the cells HIV targets. Our mini-brain model is called ’microgliacontaining cerebral organoid,‘ or MCO for short.”

Meth is a powerful, highly addictive stimulant that affects the central nervous system. It is one of the most abused drugs in America and is commonly used by HIV-infected individuals. Because both HIV and meth negatively affect the central nervous system, it’s important to understand the role the interaction of these two factors plays in the development of HAND.

Dr. Ho’s collaborator on this project is Wenhui Hu, MD, PhD, an Associate Professor in the Department of Pathology and Laboratory Medicine and in LKSOM’s Center for Metabolic Disease Research. Together, they will generate and validate the MCO model in the study of HIV infection of the brain in the context of interaction with meth.

If successful, their MCO model will allow for further research to understand the mechanism of HIV persistence in the brain, the impact of HIV and/or meth on the different types of brain cells, and the factors underlying the development of neurocognitive deficits associated with HIV infection and treatment.

“Our studies will be the first attempt to determine HIV-infected cell types and primary reservoir/latent cells in live, brain-like tissue,” Dr. Hu says. “Also, we will study how HIV and/or meth impair neural development and neuronal circuitry. Successful completion of this project should provide a clinically relevant in vitro brain model to study the roles of HIV infection and drugs of abuse in the pathogenesis of NeuroAIDS.”

BY MICHAEL TANENBAUM 
PhillyVoice Staff

HIV researchers at Temple University have become the first in their field to successfully remove the viral infection from the genomes of living animals, advancing progress in the use of powerful gene-editing technology to search for a potential cure in human beings. Scientists at the Lewis Katz School of Medicine built on a breakthrough reached last spring, when university researchers removed HIV-1 from human T-cells. This time, partnering with colleagues at the University of Pittsburgh, a study team led by Wenhui Hu demonstrated the ability to shut down HIV-1 replication in three different live animal models.The research, published in the journal Molecular Therapy, relies on the CRISPR/Cas9 genome editing tool, a versatile system that can identify genetic diseases and introduce corrective new genes into living organisms. Bioengineered RNA guides are used to detect and then edit viral strands of DNA. As a retrovirus, HIV has proven resistant to cures because it introduces replicating copies of itself into the genomes of its host cells. Current antiretroviral drugs can contain the virus and prevent the onset of AIDS, but how to permanently eradicate the infection is still an elusive goal for medical researchers. Last year, Dr. Hu's team performed a series of proof-of-concept experiments using transgenic mouse and rat models. After introducing HIV-1 DNA into the genome of every tissue in each animal's body, they were able to demonstrate the ability to remove such genetic fragments using CRISPR/Cas9. In the new study, three different models tested transgenic mice with HIV-1, mice acutely infected with their own equivalent EcoHIV infection, and "humanized" mice with latent HIV-1 placed inside engrafted human immune cells. "Our new study is more comprehensive," Dr. Hu said. "We confirmed the data from our previous work and have improved the efficiency of our gene editing strategy. We also show that the strategy is effective in two additional mouse models, one representing acute infection in mouse cells and the other representing chronic, or latent, infection in human cells." In the first model, the RNA expression of viral genes was reduced 60 to 95 percent as shown in the previous study. In the EcoHIV mice, excision efficiency reached 96 percent, marking the first evidence that this method can eradicate the virus. The third model took just one CRISPR/Cas9 treatment to excise viral fragments of latently infected human cells that were embedded in the mice. "The next stage would be to repeat the study in primates, a more suitable animal model where HIV infection induces disease, in order to further demonstrate elimination of HIV-1 DNA in latently infected T cells and other sanctuary sites for HIV-1, including brain cells," said Dr. Kamel Khalili, a lead researcher who spearheaded the study behind last year's breakthrough. "Our eventual goal is a clinical trial in human patients."

A technology developed by researchers at Temple University School of Medicine has been selected among the top 100 science stories for 2014 by 'Discover' magazine.