In vivo transfection allows for the delivery of DNA, RNAi, or mRNA molecules into live organisms and can be performed using either viral or nonviral methods. This application is considered to be more complex than in vitro transfection. However, in vivo transfection is rapidly emerging as a pivotal application in basic and clinical research as well as in the development of gene therapies.
Viral methods of in vivo transfection involve the use of engineered viral vectors (e.g., lentiviruses or adenoviruses) to deliver nucleic acids into the cells of live organisms. These viral vectors are widely used in cell and gene therapy due to their high efficiency and their capacity to integrate into the host genome for sustained gene expression (i.e., stable transfection).
However, there are several drawbacks to using viral-mediated methods, including immunogenicity, the risk for mutagenesis, and limitations to packaging capacity. In addition, viral vectors can be technically challenging and laborious to produce.
Nonviral lipid-based in vivo transfection has emerged as an invaluable means for the delivery of therapeutic agents into live organisms. Specifically, lipid nanoparticles (LNPs) are a breakthrough mechanism for performing in vivo gene delivery more safely and effectively.
The positive charge of LNPs allow them to form complexes with the nucleic acid payload (e.g., siRNA molecule). These complexes can then be injected into the organism where they enter cells via endocytosis. Once inside the cell, the nucleic acid is released by the endosome and can go on to perform its intended function (Figure 1). Of note, some LNPs consist of ionizable lipids, which exhibit a neutral charge at physiological pH that makes them less toxic to organisms than cationic lipids .
Figure 1. Overview of in vivo transfection using Invivofectamine 3.0 reagent. LNP-siRNA complexes are injected into the organism, effectively delivering the RNAi payload to target cells.
In vivo gene delivery has become invaluable to both basic and clinical research, particularly for vaccine development and in cell and gene therapy. Explore examples of specific applications below.
In vivo transfection is critical to the development of vaccines, which can use either viral or nonviral methods to deliver antigen-encoding nucleic acids into cells. LNP-mediated mRNA delivery has proven to be an especially powerful application for the development of vaccines targeting multiple diseases, including COVID-19. In addition, research into the use of personalized mRNA vaccines to target cancer cells highlights the impact and breadth of LNP-mediated in vivo transfection .
In vivo transfection has emerged as a highly promising mechanism for the replacement or suppression of disease-causing mutant genes. For example, an FDA-approved gene therapy treatment for spinal muscular atrophy (SMA) uses adeno-associated viral (AAV) vectors to deliver functional SMN1 gene copies into the motor neurons of patients .
LNPs can also be modulated to target specific organs. For example, Invitrogen Invivofectamine 3.0 reagent has been studied in animal models as a mechanism of targeted in vivo RNAi delivery for the treatment of liver fibrosis .
In vivo delivery of TALENs or CRISPR-Cas9 molecules can facilitate gene editing in live organisms. TALENs are proteins that consist of highly specific DNA binding domains and effector nucleases for site-specific cleavage, whereas CRISPR-Cas9 complexes use guide RNAs (gRNAs) that bind certain DNA sequences to allow cleavage at the site by the Cas9 nuclease. Cellular repair of cleavage sites caused by TALENs or CRISPR-Cas9 can facilitate gene silencing (i.e., via introduced mutations) or gene editing (i.e., via the insertion of a DNA donor sequence). The correction of mutant alleles for rare genetic disorders such as Duchenne muscular dystrophy in animal models highlights the significant impact of in vivo gene editing in clinical research .
Although in vitro studies are fundamental for understanding gene function, it is essential that these findings also be validated in live organisms. Therefore, in vivo transfection is a vital step in ensuring that more physiologically relevant results are obtained. Examples of transfection used in studies of gene function include introducing DNA plasmids or mRNA to examine the effects of overexpressing certain genes in organisms or using RNAi molecules to examine the effects of gene knockdown.
Invitrogen Invivofectamine 3.0 reagent from Thermo Fisher Scientific is a breakthrough product for in vivo RNAi delivery with demonstrated success of gene knockout in the mouse liver.
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For viral-mediated in vivo transfection, learn about options for lentiviral and AAV vector production.
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