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David Liu's team from the Broad Institute in the United States published a research paper titled "Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo" in the journal Nature Biotechnology. This study developed prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. The researchers systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to PE-eVLP constructs based on previously reported base editor eVLP architectures. And achieved in vivo base editing and restored partial visual function in two mouse models of genetic blindness.
The downstream processes of AAV production mainly include: cell lysis and clarification, viral vector purification (capture purification, fine purification, ultrafiltration concentration, etc.), formulation/canning, and quality testing.
Gene therapy was born to treat genetic diseases, but with the development of technology, in recent years it is going beyond the scope of treating genetic diseases and gradually entering the field of major non-genetic diseases, and even the field of solid tumors. As the field of AAV gene therapy grows, its upstream and downstream production links also need to be improved. The entire industrial production process of AAV gene therapy products can be roughly divided into three parts: upstream culture, downstream purification and finished product packaging. Among them, upstream culture and downstream purification involve more production steps and the technology is more complex. Next, let us go into the upstream of AAV industrial production to find out!
Gene therapy is one of the hottest therapeutic fields at present, and multiple gene therapies have been approved by regulatory agencies in recent years. A number of pipelines that have entered late-stage clinical studies are also approaching approval for marketing. At present, most gene therapy applications still use viral vectors. These viral vectors have been modified so that they are no longer pathogenic. Widely used viral vectors include adenoviral vectors (ADV), lentiviral vectors (LV), herpes simplex virus vectors (HSV), poxvirus vectors (PV) and adeno-associated virus vectors (AAV).
AAV has broad application prospects as a vector. In addition to being used for the treatment of genetic diseases, it can be used for the treatment of tumors and can also be used as a vector to construct vaccines. AAV also has certain applications in influenza prevention. AAV-mediated antibody expression can protect elderly and immunodeficient mice from influenza virus damage.
β2-microglobulin (B2M) is an important component of MHC class I molecules that can present tumor antigens to T cells. Loss of B2M leads to acquired tumor resistance to immune checkpoint blockade (ICB) therapy. However, there are now well-documented cases of B2M-inactivated tumors responding to ICB, which may demonstrate how the body's anti-tumor immune response is generated in tumors that do not have MCH class I molecules on their cell surfaces.
Researchers from Aarhus University in Denmark published a research paper titled "Enhanced production of mesencephalic dopaminergic neurons from lineage-restricted human undifferentiated stem cells" in the journal Nature Communications. This study constructed lineage-restricted undifferentiated stem cells (LR-USCs) by knocking out the transcription factors GBX2 and CDX1/2/4 in human pluripotent stem cells. These stem cells have a greater ability to generate specific nerve cells, dopaminergic neurons, and have produced stronger and longer-lasting therapeutic effects in rat models of Parkinson's disease.
Benjamin Kleinstiver's team at Harvard Medical School and Massachusetts General Hospital published a research paper titled "Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy" in Nature Biomedical Engineering.
Professor James Wilson's team from the University of Pennsylvania published a research paper titled "Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration" in the journal Nature Biotechnology. On the same day, Professor James Wilson's team also published a research paper titled "Prevalent and Disseminated Recombinant and Wild-Type Adeno-Associated Virus Integration in Macaques and Humans" in the journal Human Gene Therapy.
The phenomenon of RNA interference (RNAi) is triggered by double-stranded RNA that is homologous to the target gene sequence and is a post-transcriptional silencing process of sequence-specific genes that widely exists in organisms. The endonuclease Dicer in the cytoplasm cleaves dsRNA into siRNA consisting of 21-25 nucleotides, and then the siRNA combines with proteins in the body to form the RNA-induced silencing complex RISC. RISC specifically binds to the homologous region of mRNA expressed by exogenous genes and cleaves the mRNA at the binding site. After being cleaved, the fragmented mRNA is immediately degraded, thereby blocking the post-transcriptional gene silencing mechanism of the corresponding gene expression.
