Gene therapy without genes
Virus-like particles without DNA for gene therapy
Maria Moshareva, PCR.news
Scientists from the USA have created virus-like particles carrying ribonucleoproteins for editing bases instead of DNA. Their main advantage is a very low level of non—targeted editing, and the efficiency is comparable to that achieved with the use of adeno-associated vectors or lipid nanoparticles carrying mRNA. The editing partially restored the vision of mice with genetically determined blindness.
The existing variants of gene therapy use adeno-associated viruses or lipid nanoparticles carrying mRNA. The guide RNA and the editing protein are expressed in excessive amounts in the cell, and this increases the risk of inappropriate genome editing. In addition, when using viruses for gene therapy, a certain risk remains: viral DNA can be embedded in the host genome and cause oncogenic degeneration. Researchers from the Brody Institute led by David Liu and colleagues from the University of California at Irvine and the Perelman School of Medicine at the University of Pennsylvania propose to reduce these risks by using virus—like particles for gene therapy that do not contain DNA, but carry ready-made editing tools - a base editor protein and guide RNA.
Base editors are genetic tools based on a modified Cas9 protein that make it possible to make single—nucleotide substitutions in the genome without making a break in the DNA. Compared to CRISPR-Cas9, this method reduces the likelihood of accidental deletions and insertions, and therefore it is preferable in cases where genetic editing in gene therapy involves correcting a point mutation.
The researchers assembled the components of the base editor system in HEK293T cells into a virus-like particle based on retroviruses, and then used such ready-made particles to edit cells ex vivo and in vivo. The sequence of the base editor is fused with the sequence of the MLV viral protein, this ensures the packaging of the target protein in complex with the guide RNA into the viral capsid. The specificity of virus-like particles to a specific cell type is achieved through the use of different packaging glycoproteins.
The main problem faced by the authors is the packaging of virus—like particles and the editing of DNA in target cells put forward different requirements for the cellular localization of the base editor protein. For successful editing, it is desirable that the base editor has a sequence of nuclear localization, but with nuclear localization, protein packaging into a virus-like particle is impossible.
The authors applied a dual localization system to solve this problem. An export signal from the nucleus was added to the sequence of the viral protein, and the sequence of the base editor was flanked by nuclear localization signals. Between them is the site of cutting by a protease, which is included in the composition of a virus-like particle. Thus, the particle is assembled in the cytoplasm, and in the target cell, a split fragment of the base editor carrying nuclear localization signals is sent to the nucleus.
The system was tested on HEK293T cells, while the editing efficiency was comparable to that achieved with conventional transfection, but the frequency of non-targeted editing was reduced by 12-900 times. The efficiency of editing primary cell cultures was also close to 100%, non-targeted editing was also relatively rare.
When virus-like particles were injected into the cerebrospinal fluid of newborn mice, the efficiency of editing the Dnmt1 gene in the cells of the volumetric cortex and midbrain was 6.1% and 4.4%, respectively. When editing the therapeutically significant Pcsk9 gene in adult mouse liver cells, the efficiency reached 63% — a result comparable to alternative approaches, and at the same time the level of non-targeted editing turned out to be undetectable.
The authors also applied a new approach to the treatment of mice with a model of genetically determined blindness caused by a mutation in the Rpe65 gene (Leber congenital amaurosis). The editing efficiency was 12%, and after a single injection, the animals' vision partially recovered.
As a possible limitation of the method, the authors call the non-targeted capture of random cell components into the composition of a virus-like particle, which may entail difficult to predict risks.
Article by Banskota et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins is published in the journal Cell.
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