What technologies do nucleic acid drug companies such as Moderna use?
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What technologies do nucleic acid drug companies such as Moderna use?
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What technologies do nucleic acid drug companies such as Moderna use?
What technologies will be widely adopted by leaders in RNA medicine? Such as Moderna, BioNtech and CureVac in the field of mRNA drugs, Ionis Pharmaceuticals, Sarepta, etc. in the ASO field, Alnylam, Dicerna and Arrowhead in the siRNA field, MiNA Therapeutics in the saRNA field, Zhongmei Ruikang, etc., and various other RNA therapy companies.
With the success of the mRNA COVID-19 vaccine, RNA drugs have entered a stage of rapid development.
On a global scale, RNA therapeutics provide a unique opportunity for researchers in various countries to expand the scope of drug development targets.
There are many different types of RNA therapy in the development process, mainly including antisense oligonucleotide (ASO), small interfering nucleic acid (siRNA), microRNA (miRNA), messenger RNA (mRNA), aptamer (nucleic acid aptamer) and many more.
As a new type of drug therapy, it can selectively act on proteins and genes that cannot be targeted by conventional drug molecules. RNA nucleic acid drugs have been used in many different diseases.
Despite extraordinary progress in RNA therapy R&D, there are still many problems and challenges to be overcome, such as high RNA instability, low in vivo delivery efficiency, host cell immunogenicity, etc.
RNA molecules are directly used in two modalities of therapy in the form of drugs or targeted RNA small molecules.
RNA drugs can utilize endogenous regulatory mechanisms and can specifically modulate their target targets.
RNA drugs must pass through the cell membrane and have the ability to bind intracellular targets and regulate gene expression, thereby achieving regulation of related protein expression.
Fundamentals of various RNA therapeutics
The detection of protein expression level is a very important aspect of pharmacological and pharmacodynamic research.
Currently, there are various techniques for quantitative analysis of secreted and endogenous proteins in different matrices, such as ELISA technology, LC-MS technology, Western Blot technology, etc.
There are also some Innovative technologies are gradually being adopted by mainstream nucleic acid drug companies.
This article takes a look at what technologies are used by nucleic acid drug leaders to solve protein analysis problems in research and development.
01 Moderna’s protein expression quantification technology in preclinical animal models
Moderna is currently developing a clinical phase I mRNA therapy, mRNA-3927 for the treatment of propionic acidemia (PA).
Preclinical mouse animal model studies have shown that dual RNA therapy can restore metabolic function.
Propionic acidemia is caused by deficiency of a mitochondrial enzyme, propionyl-CoA carboxylase (PCC), which is composed of six alpha (PCCA) and six beta (PCCB) subunits.
Using a dual mRNA combination encoding human PCCA and PCCB loaded in biodegradable lipid nanoparticles (LNPs), a functional PCC enzyme was produced in the liver.
Proteins encoded by double mRNAs have higher PCC enzymatic activity than single mRNAs. Bottom panels a-c are data comparing double RNA co-transfection versus hPCCA or hPCCB mRNA transfection alone.
Panels a and d are assessments of enzymatic activity, panels b and c are quantitative detection of PCCA and PCCB protein expression in patient fibroblasts using capillary Digital Western.
02 Quantitative detection of hOTC expression in Arcturus Therapeutics tissue
Arcturus Therapeutics’ clinical-stage mRNA therapy ARCT-810 is indicated for the treatment of Ornithine Transcarbamylase Deficiency (OTCD).
The disease is the most common inherited metabolic disease in urea cycle disorders, and mutations in the OTC gene can cause hyperammonemia episodes.
Lipid nanoparticle (LNP)-loaded OTC mRNA (LUNAR-OTC mRNA) utilizes proprietary lipid drug-mediated technology to safely and efficiently deliver OTC mRNA into hepatocytes, enabling natural production in the patient’s own hepatocytes A functioning OCT enzyme, in turn.
In the preclinical mouse model study, the efficacy of LUNAR-OTC mRNA drug in the mouse model was reflected by measuring the OTC enzyme activity and protein expression in the liver and plasma of mice treated with OTC-mRNA drug.
Protein expression of human ornithine transcarbamylase (hOTC) in mouse liver.
The expression of hOTC protein in mouse liver was quantitatively detected by fully automatic capillary Digital Western (Jess Simple Western) system, and the recombinant protein was used as the calibration curve for quantification.
Using anti-human OTC antibodies, a clear dose response was observed from the four escalating dose groups, with no hOTC protein detected in either the untreated OTC-deficient group or the untreated wild-type group.
03 Science sub-journal: ASO inhibition of RBM20 improves heart failure function
Heart failure with preserved ejection fraction (HFpEF) is common and fatal, and so far, there are no well-targeted drugs.
One of the etiologies is impaired ventricular filling, and the use of antisense oligonucleotides (ASOs) targeting the cardiac splicing factor RBM20 improves its function.
In adult mice with increased wall stiffness, weekly administration of ASO for 2 months increased compliant actin isoform expression and improved cardiac function.
Validation results in human engineered heart tissue showed that RBM20 expression was downregulated to less than 50% within 3 weeks of ASO treatment, resulting in adaptive relaxation kinetics in the absence of cardiac pathology.
This study demonstrates that anti-RBM20 ASO acts as a powerful cardiac splicing regulator in the treatment of human HFpEF.
Injection of RBM20 ASO effectively inhibited the expression of RBM20 in mouse heart.
(B) is the experimental design in mice,
(C) Rbm20 mRNA expression in heart tissue of wild-type (WT) and N2B knockout (KO) mice treated with PBS or ASO,
(D and E) using fully automatic Corresponding RBM20 protein expression abundance was detected by capillary Digital Western.
At the molecular level of mRNA and protein expression, the expression abundance of RBM20 was down-regulated by more than 50%.
Fully automatic capillary Digital Western technology is an advanced tool for protein expression quantification in nucleic acid drug development
The above three cases of Moderna, Arcturus Therapeutics and Ionis Pharmaceuticals are only representative cases of Digital Western, and this technology has been widely adopted by leaders in the field of RNA drugs.
Such as Moderna, BioNtech and CureVac in the field of mRNA drugs, Ionis Pharmaceuticals, Sarepta, etc. in the ASO field, Alnylam, Dicerna and Arrowhead in the siRNA field, MiNA Therapeutics in the saRNA field, Zhongmei Ruikang, etc., and various other RNA therapy companies.
As an innovative capillary electrophoresis immunology technology, the fully automatic capillary Digital Western technology combines Western Blot technology and ELISA technology into one, and can achieve accurate relative and absolute quantification of endogenous proteins by using trace tissue samples.
As an innovative biological analysis method, it has the advantages of traditional Western Blot protein molecular weight visualization, more accurate qualitative, and accurate quantitative performance of ELISA and MS technology, and its sensitivity and precision meet the requirements of high standard biological analysis.
Technical advantages for RNA therapy applications
1) Digital Western technology is suitable for quantitative analysis of target protein expression in various models of RNA therapy in vitro and in vivo, for key protein analysis in cell lines, organoid models, mouse animal models and non-human primate models.
2) The sample volume requirement of Digital Western technology is a few tenths of that of traditional Western Blot, and multiple protein expression detection can be realized with only 3 μL of sample, which is especially suitable for protein analysis of trace precious samples.
3) Digital Western accurate quantitative detection, traditional Western Blot can only meet the semi-quantitative requirements of samples, and the repeatability is relatively poor.
The expression of some target proteins of RNA therapy is correlated with clinical treatment effect and can be used as a surrogate biomarker to establish a dose-response relationship.
The detection standard of target protein analysis needs to be improved, and the technology needs to be rigorously validated, and Digital Western can meet these needs.
4) In line with the needs of RNA therapy for automation standardization and efficiency, in the face of fierce competition in the industry, it is necessary to improve the efficiency of research and development.
Digital Western is fully automated and standardized, and the software meets FDA 21 CFR Part 11 compliance requirements.
The system completes a batch of protein analysis in 3 hours, which is faster than traditional Western Blot and ELISA, greatly improving experimental efficiency and reducing labor costs.
What technologies do nucleic acid drug companies such as Moderna use?
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