Disadvantages of the In-Vivo Transfection 

• The unexpected immune system can generate in the body as the result of the in-vivo transfection.  
• In-vivo transfection or gene transfer can initiate or activate any viral protein or activate the disease-causing pathogens inside the body.  
• In-vivo transfection can cause tumors in the body, which is related to the abnormal growth of the cells. This abnormal growth of the cells can disrupt the body’s functioning.  
• Scientists can also make a mistake, and there is the possibility that scientists unwantedly target one or more genes or cells, which can lead to the failure of the technique.  

Clinical Scope of the In-Vivo Transfection 

In-vivo transfection is helpful in research or the medical field. Scientists implemented this technique to treat some malignancies and are doing continuously successful trials. In this method, scientists affect the target cells only, not the whole body Applications for treating human diseases are increasingly evolving, and researchers are silencing the genes or which genes fail to express. They are increasing the expression of genes. However, the strategies and the efficacy are highly variable from organism to organism.   

Types of Transfected Nucleic Acids 

The transfection process involves delivering foreign nucleic acids into eukaryotic cells to modify their genetic makeup. The application of transfection to studying cellular processes and molecular mechanisms of disease has gained increasing popularity over the past 30 years. Listed below are all the types of nucleic acids that can be transfected. 

• Deoxyribonucleic acids 

Viral or non-viral vectors such as plasmids are normally used to transfect DNA into a host cell. Basic plasmid structures include a promoter, replication origin, multiple cloning sites, gene of interest, and selection marker. It is necessary to have the origin of replication to replicate plasmids, while the multiple cloning site contains unique restriction enzyme cut sites that allow foreign genes to be inserted.  

EF-1* or CMV promoters can promote the expression of a foreign gene in eukaryotic cells. It is possible to transfect plasmid DNA in either linear or supercoiled forms. A supercoiled plasmid generally has a higher transfection efficiency than linear DNA, which is more vulnerable to exonuclease degradation. On the other hand, the linearized DNA is more recombinogenic and, therefore, has a higher chance of integrating into the host genome to achieve stabilization. 

It is not guaranteed that plasmid vectors will result in constitutive transgene expression or that foreign DNA will be stably incorporated into the host genome. If a foreign gene has not been integrated into the host cell genome, it will not be constitutively expressed and eventually be lost.  

To maintain stably transfected cells in culture, appropriate selection markers, such as antibiotic resistance genes or fluorescence proteins, must be used. Virus vector transfection is also useful for selecting transduced cells that are stably transduced by introducing a selection marker gene. 

Transfection with plasmids is less immunogenic than viral DNA transfection since there is no risk of viral integration into the host cell’s genome. However, plasmid-based DNA transfection has a lower transfection efficiency and protein production. 

• RNA And Messenger RNA 

Like DNA, it is also possible to introduce RNA into eukaryotic cells via RNA-based viral or non-viral vectors. As RNA transfection does not require transit across the nuclear membrane, it might produce higher transfection efficiency than transfection with DNA. The transfection of RNA can also accelerate the production of the desired protein since no genome integration, transcription, or post-transcriptional processing is required.  

Vectors based on messenger RNA (mRNA) can also avoid complications caused by integration into the host genome, allowing specific, desired proteins to be expressed. In contrast to DNAs, RNAs are comparatively less stable and prone to degradation when transported intracellularly, resulting in transient protein expression following RNA transfection. 

• Common And Special Oligonucleotides (small ribonucleic acids) 

A small RNA is a molecule of RNA consisting of 18–200 base pairs (bp) and capable of regulating post-transcriptional gene regulation. In addition to microRNAs (miRNAs), siRNAs (small interfering RNAs), short hairpin RNAs (shRNAs), and piRNAs (piRNAs) are examples of small RNAs. 

MicroRNAs, piRNAs, And shRNAs 

There are two types of small RNAs, microRNAs, and piRNAs, both endogenous, single-stranded RNAs. miRNAs (18–25 bp) inhibit or interfere with the translation initiation of downstream mRNAs through post-transcriptional regulation. At the same time, piRNAs (24–30 bp) play a role in post-transcriptional regulation and transposon silencing.  

A siRNA plays a similar role in post-transcriptional gene regulation as miRNAs and piRNAs. The length of siRNAs is normally 20–24 bp, which can be expressed either endogenously or exogenously. Small RNAs with hairpin loops, called shRNAs, are endogenous, double-stranded RNAs. As a result of binding to the complementary sequence on an mRNA, shRNA can degrade it. It is important to determine the experimental need before choosing a small RNA molecule for transfection-related functional assays. 

In contrast to siRNA, miRNA can regulate multiple downstream targets while being highly specific to only one. For functional studies of the knock-in, down, and out effects of these small RNA molecules, a variety of synthetic short-length oligonucleotides can be synthesized artificially to mimic small RNA molecules.  

• Mimics and Antagonists 

There are two types of oligonucleotides: mimics and antagonists. A mimic is a small RNA-based oligonucleotide with a structure that can bind to targeted mRNAs to inhibit their function, thereby inhibiting translation. A complimentary small RNA strand, such as miRNA, can be victimized by an antagonist oligonucleotide, thereby increasing the expression of the targeted gene. 

Different types of modified oligonucleotides were also introduced into the market with the advancement of oligonucleotide biosynthesis to increase the efficiency of transfecting small RNA oligonucleotides. 

Chemically modified agomirs and antagomirs have enhanced binding affinities to target and block exonuclease activity. Compared to conventional miRNA mimics, agomirs are artificially modified double-stranded miRNAs that exert higher target repression activity.  

In contrast, antagomir targets a specific miRNA and is specifically designed to inhibit it. As compared to the normal mimics or antagonists, agomir and antagomir were claimed to be more stable, highly effective, and specific. 

• Locked Nucleic Acids (LNAs)  

Locked nucleic acids (LNAs) contain an extra methylene bridge in at least one nucleotide, enhancing their stability as ribose ring structures in another type of modified oligonucleotide. As a result of LNA’s locked ribose structure, it has higher efficiency, stability, and binding affinity than traditional oligonucleotides.  

Small RNA molecules such as siRNA, miRNA, and piRNA have been delivered with LNA-based oligonucleotides in various biochemical and functional assays. Some LNA-based transfections don’t require transfection reagents, minimizing the effects of the reagents during transfection.