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  • OVERVIEW
Antibody Production by mRNA Immunization
  • OVERVIEW

Antibody Production by mRNA Immunization


Seattle Genova offers antibody production services by mRNA immunization. Our unique strategy can help our customers to develop antibodies that are targeting the natural configuration of an antigen. Such antigens can be, viral envelope proteins, membrane proteins and some other difficult-to-prepare proteins. Basically, immunogen sequences are designed and are inserted into a DNA vector template, and their sequences were further encoded into mRNA molecules by in vitro transcription, the mRNAs are encapsulated in lipid nanoparticles and delivered by infusion into mice or rabbits.


mRNAs are transient blueprints of genes that are encoded in the genomic DNA. The mRNA transcripts of these genes deliver the genetic information to the translational machinery to generate the encoded proteins. In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases. The template for in vitro transcription of mRNA consists of five in cis-acting structural elements, namely from 5′ to 3′ end: (i) the optimized cap structure, (ii) the optimized 5′ untranslated region (UTR), (iii) the codon optimized coding sequence, (iv) the optimized 3′ UTR and (v) a stretch of repeated adenine nucleotides (polyA tail).


Advantages of mRNA immunization

1. -mRNA is precise as it will only express a specific antigen and induce a directed immune response.

2. -It promotes both humoral and cellular immune response and induces the innate immune system.

3. -Compared with DNA-based immunization, mRNA is more effective, since expression does not require nuclear entry, and safer, since the probability of random genome integration is virtually zero.

4. -Expression of the coded antigens is transient since mRNA is quickly degraded by cellular processes, with no traces found after 2–3 days. Therefore, multiple immunizations can be done in 28 days.

5. -Since production is based on an in vitro cell-free transcription reaction, concerns regarding the presence of cell-derived impurities and viral contaminants commonly found in other technologies are minimized.

6. -Most importantly, the mRNA encoded proteins are expressed either on the cell surfaces or inside of the cytosols thus they are in the natural configurations which can help to generate antibodies that are specific to native antigens.


Self-replicating RNA

Another RNA-based approach to immunization is self-amplifying RNA immunogens. The backbone sequence for the latter is adapted from an alphavirus, a positive-sense single-stranded RNA virus with high capacity for replication. Such mRNA vaccine contains an antigen-encoding sequence and viral RNA dependent RNA polymerase-encoding sequence along with other elements required for replication. The advantage of the self-replicating approach is that significantly higher amount of antigen can be expressed with lower doses of mRNA. Both types of RNA vaccines degrade after transient expression; however, the self-replicating RNA achieves longer term expression of the antigen.


Required antigen preparation

In vitro transcribed mRNA immunogens must be prepared in advance of the immunization and fusion protocols. We offer mRNA design, in vitro transcription, and mRNA purification services. The amounts of materials that must be prepared in advance of each protocol are as follows:

·         4 mg mRNA in total

·         2 mg purified antigen or synthesized peptides for the screening step

·         Antigen-expressing stable cell lines are recommended for the screening as well

Antibody Production by mRNA Immunization


Seattle Genova offers antibody production services by mRNA immunization. Our unique strategy can help our customers to develop antibodies that are targeting the natural configuration of an antigen. Such antigens can be, viral envelope proteins, membrane proteins and some other difficult-to-prepare proteins. Basically, immunogen sequences are designed and are inserted into a DNA vector template, and their sequences were further encoded into mRNA molecules by in vitro transcription, the mRNAs are encapsulated in lipid nanoparticles and delivered by infusion into mice or rabbits.


mRNAs are transient blueprints of genes that are encoded in the genomic DNA. The mRNA transcripts of these genes deliver the genetic information to the translational machinery to generate the encoded proteins. In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases. The template for in vitro transcription of mRNA consists of five in cis-acting structural elements, namely from 5′ to 3′ end: (i) the optimized cap structure, (ii) the optimized 5′ untranslated region (UTR), (iii) the codon optimized coding sequence, (iv) the optimized 3′ UTR and (v) a stretch of repeated adenine nucleotides (polyA tail).


Advantages of mRNA immunization

1. -mRNA is precise as it will only express a specific antigen and induce a directed immune response.

2. -It promotes both humoral and cellular immune response and induces the innate immune system.

3. -Compared with DNA-based immunization, mRNA is more effective, since expression does not require nuclear entry, and safer, since the probability of random genome integration is virtually zero.

4. -Expression of the coded antigens is transient since mRNA is quickly degraded by cellular processes, with no traces found after 2–3 days. Therefore, multiple immunizations can be done in 28 days.

5. -Since production is based on an in vitro cell-free transcription reaction, concerns regarding the presence of cell-derived impurities and viral contaminants commonly found in other technologies are minimized.

6. -Most importantly, the mRNA encoded proteins are expressed either on the cell surfaces or inside of the cytosols thus they are in the natural configurations which can help to generate antibodies that are specific to native antigens.


Self-replicating RNA

Another RNA-based approach to immunization is self-amplifying RNA immunogens. The backbone sequence for the latter is adapted from an alphavirus, a positive-sense single-stranded RNA virus with high capacity for replication. Such mRNA vaccine contains an antigen-encoding sequence and viral RNA dependent RNA polymerase-encoding sequence along with other elements required for replication. The advantage of the self-replicating approach is that significantly higher amount of antigen can be expressed with lower doses of mRNA. Both types of RNA vaccines degrade after transient expression; however, the self-replicating RNA achieves longer term expression of the antigen.


Required antigen preparation

In vitro transcribed mRNA immunogens must be prepared in advance of the immunization and fusion protocols. We offer mRNA design, in vitro transcription, and mRNA purification services. The amounts of materials that must be prepared in advance of each protocol are as follows:

·         4 mg mRNA in total

·         2 mg purified antigen or synthesized peptides for the screening step

·         Antigen-expressing stable cell lines are recommended for the screening as well

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