Tag Archives: mRNA therapeutics

How do we make 2 successful COVID mRNA vaccines?

Introduction

According to COVID data from World Health Organization (2022/02/28), total COVID cases have reached 400 million, and the COVID-induced death is close to 6 million people. On the bright sight, the COVID vaccines have been administered over 10 billion doses, and around 4 billion people are fully vaccinated now. Among the FDA-approved COVID vaccine, the primary modality is mRNA-based vaccines from Moderna or Pfizer/BNT. In February 2022, Moderna announced establishing subsidiaries in four Asian countries, including Taiwan, and planning to manufacture COVID vaccine and other mRNA-based drugs in Taiwan. What is the procedure to make an mRNA vaccine and mRNA-based drugs? Here I will explain how the lipid nanoparticles with mRNA are manufactured.

Figure 1, the mechanism of gene therpay: DNA transcripts into RNA which could translate into proteins in cells.

Mechanism of gene therapy

First, we need to know what mRNA is. In our cells, chromosomes composed of DNA and proteins in the nucleus control the gene expression by different DNA fragments. Additionally, the mechanism is that first, the DNA in the nucleus transcripts into messenger RNA (mRNA), and the mRNA would translate into protein to be expressed in the cells (Figure 1 ). Also, that’s why mRNA plays an essential role in molecular biology. To produce the mRNA, we would need a lot of DNA templates for in vitro transcription (IVT), and the easier way to collect a large number of DNA templates is to clone the desired DNA template into a plasmid and transforms it into E. coli, to use E. coli to grow and produce a lot of plasmids for IVT. After collecting the plasmids from E. coli, we would use enzymes to isolate the DNA template to make the RNA through IVT reactions. (Figure 2) The mRNA structure comprises the RNA coding region, untranslated region, capping, and poly-A tail. (Figure 3) Therefore, we need to add 5′ capping and 3′ poly-A tail to the IVT RNA before purification. The mRNA production is a simple reaction, but several essential elements affect the mRNA efficacy.

Key elements in IVT reaction

First, we need to add a T7 promoter in the DNA template because T7 RNA polymerase is required in the IVT. Second, the RNA coding region should be designed by codon-optimization to prevent the single-strand RNA from forming secondary structure to decrease the efficacy. Third, there is an untranslated region (UTR) on each side of the coding region. Although the UTR would not be translated into protein, these regions could affect the efficacy of mRNA in the cells. Additionally, a good UTR sequence could improve protein production in vitro and in vivo and alleviate the immune response triggered by the mRNA. In the biotech company, the UTR sequences are confidential because they could highly affect mRNA therapeutics’ efficacy. Last, the 5′ cap is also a crucial element in mRNA synthesis. In the COVID vaccine from Pfizer and Moderna, the capping system, CleanCapⓇ is from another mRNA biotech company, Trilink, to minimize the immune response in the body and increase the vaccine efficacy. Therefore, the whole production of mRNA is pretty complicated. I will prepare another blog to introduce the IVT reaction and the following procedure to produce mRNA.

Figure 2, scalable production of DNA template for in-vitro transcription (IVT) reactions.
Figure 3, The mRNA structure comprises the RNA coding region, untranslated region, capping, and poly-A tail.

Lipid nanoparticles production

Next, after purification of capped mRNA with polyA tail, we could mix the mRNA and lipid-like materials to manufacture the lipid nanoparticles (LNP). In the previous blog (introduction to gene therapy), I have introduced how cationic lipid-like materials form LNP under acidic conditions by electrostatic interaction. The critical factor in LNP production is mixing because well-mixing could allow the cationic materials and anionic nucleic acids to form a more compact structure and smaller nanoparticles. To make those mRNA-based LNP with less than 100 nanometers in diameter, we used a microfluidic device with a micromixer to better mix the organic phase (lipid-like materials) and aqueous phase (nucleic acids) by chaotic advection (Figure 4). The smaller size of LNP could prevent liver filtration after administration. Furthermore, another essential step is how we purify the LNP and remove the residuals during the process to minimize the side effects of the mRNA-based vaccine.

Figure 4, microfluidic device with micromixer is used to produce lipid nanoparticles with mRNA.

Conclusion

Although the whole process looks pretty simple, every step in manufacturing is critical to maintaining a safe and efficient vaccine. There is still a lot of work we could do in research to improve the mRNA therapeutics: How we design the coding sequences UTR sequences. How we develop a new capping system and nucleotides to alleviate immune response. Also, currently, all the FDA-approved mRNA-based vaccines are used non-degradable materials to deliver mRNA. Most scientists and biotech companies are working on new degradable materials in RNA therapeutics. Therefore, if you are interested in gene therapy, maybe you could consider contributing to the field of mRNA therapeutics.

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Rejuvenation, next generation therapeutic could transform our lives in 10 years?

Aging is one of the reasons that scientists work hard to do biomedical research, however, rejuvenation is an unreachable dream. Especially in fiction movies, rejuvenation is always a key element. Is it possible to let people keep young-looking forever? How does it work? If so, how does it change the current biomedical industry or even the cosmetic industry?

The first thing I would like to introduce is “Yamanaka factors,” which brought Kyoto University researcher Shinya Yamanaka to win the Nobel prize in 2012. What are Yamanaka factors? Four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4) are the Yamanaka factors, and they could be applied to reprogram somatic cells into pluripotent stem cells. In other words, with Yamanaka factors, we could generate unlimited stem cells from people or any patients who need organ transplantation. We could think of the stem cells as age zero because every fetal is grown from embryonic stem cells, and the Yamanaka factors could induce the cells from a mature state to the original condition. This fact allows scientists to think about rejuvenation; what if we only partially reprogram the cells? Is it possible to make the cells younger? In 2020, Dr. Sinclair and his group used the adenovirus-associated virus (AAV) to deliver three Yamanaka factors to restore vision in mice, and this work has been published in Nature. Also, Dr. Vittorio Sebastiano and his group demonstrated how Yamanaka factors with two additional factors (LIN28 and NANOG) reverse the epigenetic methylation of cytosine-guanine dinucleotides (CpG) on DNA to make the human cells younger with restored regeneration. Here, I would like to explain what epigenetic methylation is and what epigenetic clock is? First, CpG islands are the region on DNA that could be added a methyl group (CH3 group), and the DNA methylation could regulate gene expressions. In this hypothesis, stem cells would have less methylation of CpG on DNA. Second, with epigenetic methylation, a new definition of age is epigenetic clock/age, calculated and measured by the change in DNA methylation and the biomarkers of age. It is different from the chronological age counting by time, but epigenetic age is measured by the methylation of DNA. An exciting discovery is that the rate of epigenetic aging is slower in supercentenarian and their descendants. This might explain why those people could live longer than others. On the other hand, previous studies give us hope that if we could use the Yamanaka factors to partially reverse epigenetic methylation frequently, we might make dreams come true that we all become centenarians and have young-looking forever.

With the potential as therapeutics, several companies have been found to develop anti-aging as rejuvenation therapy. For example, Calico Life Sciences/Alphabet, which was founded by Bill Maris and Arthur Levinson in 2013, focuses on basic research in partial reprogramming mechanisms. Life Biosciences which was found by David Sinclair and Tristan Edwards in 2017, focuses on using AAV to deliver reprogramming factor genes. Turn Biotechnologies which was found by Vittorio Sebastiano, Marco Quarta, and Jay Sarkar in 2019, focuses on mRNA-based delivery of reprogramming factors. Last, a newly launched biotechnology company, Altos Labs, which is invested by the founder and former CEO of Amazon, Jeff Bezo, focuses on transforming medicine through cellular rejuvenation programming. The company has three Altos Institutes of Science based in the San Francisco Bay Area, San Diego, and in the UK (Cambridge). In the increase in aging research and newly launched biotech companies, maybe rejuvenation is a potential therapeutic to extend average human life; however, the current research only accomplished the epigenetic reprogramming in a rodent model and human cells in vitro. I believe it will not be an easy task to reverse the epigenetic methylation in non-human primates and also in humans. The four Yamanaka factors are not enough to completely reprogram the cells in the body. Therefore, there will be a lot of things we could do in the aging research, especially after the COVID-19 pandemic, mRNA therapeutic becomes a very popular tool to deliver genes in the human body, for instance, the COVID vaccine.

In sum, rejuvenation was a myth story to us, but with accelerated development in biotechnology, anti-aging would be accomplished and improve human health in the next decade.

Reference:

  1. Michael Eisenstein, nature biotechnology 2022 https://doi.org/10.1038/d41587-022-00002-4
  2. Sarkar, T.J., Quarta, M., Mukherjee, S. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat Commun 11, 1545 (2020). https://doi.org/10.1038/s41467-020-15174-3
  3. Lu, Y., Brommer, B., Tian, X. et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature 588, 124–129 (2020). https://doi.org/10.1038/s41586-020-2975-4
  4. Chiavellini P, Canatelli-Mallat M, Lehmann M, Gallardo MD, Herenu CB, Cordeiro JL, Clement J, Goya RG. Aging and rejuvenation – a modular epigenome model. Aging (Albany NY). 2021; 13:4734-4746. https://doi.org/10.18632/aging.202712
  5. Altos Labs company website

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Jason(Yen-Chun) Lu, All right reserved.

Introduction to Gene Therapy

Recently, gene therapy has become a hot topic in the biomedical research and pharmaceutical industry because the timeline of new drug approval for the US Food and Drug Administration (FDA) is shorter than traditional drug modalities, such as small molecular drugs. In addition, after the COVID pandemic, gene-based technology plays an important role in vaccine development with its advantage in completing the clinical trial for new drug applications (IND). Taking Moderna as an example, after Chinese authorities shared the genetic sequence of the COVID, the National Institute of Health (NIH) and Moderna finalized the sequence for its mRNA product for the COVID vaccine. Moreover, Moderna began the clinical trial in April 2020 and received the emergency use of its mRNA vaccine (mRNA-1273) for COVID-19 from the FDA in December. This is a great example to show how fast gene therapy could be approved and authorized by FDA.


Before discussing the types of gene therapy, I would like to briefly introduce nucleic acid, which is an essential sugar-based biomolecule in all cells and viruses. Nucleic acids are divided into two famous classes, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These nucleic acids store pieces of information in the cells and also passage this information to our descendants. Moreover, the DNA is composed of 4 different units, Adenine (A), Thymine (T), Cytosine (C), and Guanine (G), and RNA is composed of Adenine (A), Uracil (U), Cytosine (C) and Guanine (G). DNA can transcript into RNA in the cells and then translate into protein to work for the cells. For example, the mechanism of the COVID vaccine from Johnson& Johnson or AstraZeneca is to deliver the DNA sequence of spike protein in coronavirus by adenovirus. When the DNA fragment is given in the cells, it will transcript into mRNA and translation into the spike protein. So the immune system could recognize the proteins and produce antibodies to protect the body if any coronavirus is found. In the mRNA vaccine, the mechanism is straightforward because the targeted mRNA could directly deliver to the cells and produce the proteins.

Figure to illustrate how DNA convert into RNA and translate into proteins

Up to date, there are two types of gene therapy, virus-based, and non-virus-based delivery system. In a virus-based delivery system, adenovirus and adeno-associated virus (AAV) are the primary vehicles to deliver nucleic acids. In the COVID vaccines, two companies (AstraZeneca and Johnson&Johnson) used adenovirus to deliver the DNA sequence of spike protein on coronavirus. Also, there are two FDA-approved AAV-based therapies: First, Spark Therapeutics developed Luxturna for degenerative disease of the eye (retinal dystrophy). Second, Novartis uses Zolgensma to treat spinal muscular atrophy (type I). On the other hand, the non-viral system mainly focuses on synthetic materials, such as polymeric materials, ionizable lipids, peptides, and zwitterionic lipids and dendrimers. In the early research, scientists focused on cationic materials to form nanoparticles with nucleic acids; however, researchers have recently worked on pH-dependent ionizable materials with lower toxicity. In an acidic environment, the ionizable materials become cationic and form nanoparticles with anionic nucleic acids by electrostatic force. These ionizable materials could protect the therapeutic mRNA from degradation and selectively target the specific cells for delivery. Additionally, after the lipid nanoparticles enter the cells, the mRNA would be released in a neutral cytoplasm environment where the mRNA would start to translate into protein to treat disease or build up the protection from any virus or bacteria. Since the mRNA-based vaccine for COVID, the non-viral system for mRNA delivery has become a popular field in gene therapy development.

Illustrate how COVID (AstraZeneca) vaccine works in the body


In sum, with the fast developing timeline, gene therapy would be a major modality in pharmaceutical companies in the next decade and also benefit human health welfare, such as vaccine development in different viruses.

Reference:

  1. P. Kowalski et. al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery.”, Molecular Therapy Vol. 27, No. 4, 2019
  2. S. Liu et. al., “Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.”, Nature Materials Vol. 20, 2021

Jason(Yen-Chun) Lu, All right reserved.

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What engineers can do in biomedical field?

An engineer is trained to solve problems through science and math. We can see engineers everywhere in our lives, such as civil engineers, chemical engineers, electrical engineers, and software engineers, to improve our daily lives. What can engineers do in the biomedical field? A pioneer, professor Robert Langer, an internationally well-known biomedical engineer and inventor, is a perfect example illustrating how engineers can contribute to biomedical science. Dr. Langer holds the title of David H. Koch Institute Professor at the Massachusetts Institute of Technology (MIT) as well as that of Senior Lecturer on Surgery at Harvard University’s Medical School. It is worth to mention that being an Institute Professor at MIT is the highest honor that can be awarded to a faculty member. Besides, Dr. Langer has more than 1,500 published papers and over 1,400 issued patents and pending patents worldwide. In his research, he focuses on solving biomedical problems from an engineering aspect, such as developing materials for drug delivery, cell engineering, and tissue engineering. Here we have some examples to demonstrate how engineers contribute to the field.

First, Dr. Langer and his colleagues with Bill & Melinda Gates Foundation created pulsatile-release PLGA microspheres for single-injection vaccination1 for developing world. Poly (lactic-co-glycolic acid) (PLGA) is an FDA-approved degradable material for clinical application, and core-shell decoupled microspheres are fabricated by a new microfabrication method (StampEd Assembly of polymer Layers (SEAL))2. Despite the immense increase in vaccine coverage worldwide over decades, vaccine-preventable infectious diseases still claim the lives of approximately 1.5 million children every year because of inadequate distribution and administration of vaccines in the developing countries. Currently, around 19.4 million infants do not receive fully immunized against diphtheria, tetanus, and pertussis. Moreover, 6.6 million of them with one dose of the vaccine remain at risk for these diseases due to lack of full series of doses. With the pulsatile-release PLGA microspheres and SEAL technology, the problem of inadequate distribution and administration of vaccine could be solved, and millions of people in the developing world would benefit.

What engineers can do in biomedical field? 1

Fig. 1, Using different molecular weight of PLGA to control degradation time to release the therapeutics to evoke immune response. (modified from McHugh, K. J. et al. Science, 2017).

Second, Dr. Langer and his colleagues discovered three chemical materials which can suppress foreign body response to minimize fibrosis in rodents and at least 6 months in non-human primates3. These materials were conjugated to alginate hydrogel, and these hydrogel microspheres were transplanted in mice and monkeys. In addition, these anti-fibrotic materials could be applied in cell therapy, such as beta cell replacement treatment for type I diabetes. In type I diabetes, patients’ pancreatic islet cells are destroyed by their own immune system. To date, the most common treatment is a daily insulin injection to control blood glucose. However, insulin injection cannot cure type I diabetes or prevent the many devastating diseases associated with diabetes, such as blindness, hypertension, and kidney disease. Islet cell transplantation could provide an alternative treatment for type I diabetes to avoid daily injection and restore normoglycemia. However, foreign body response is a major challenge for cell therapy. The cellular and collagenous deposition would isolate the transplanted device from the host, which could induce tissue distortion, cut off the nourishment of encapsulated cells, and finally lead to device failure. With these new anti-fibrotic materials, the transplanted device with insulin-producing beta cells could maintain its function in the long term to cure type I diabetes.

What engineers can do in biomedical field? 2

Fig. 2, Three chemical materials can suppress foreign body response to minimize fibrosis in rodents and non-human primates. Encapsulated by these materials, the therapeutic cells can be protected from host immune system and also suppress its immune system to reduce foreign body response.

Third, Dr. Langer’s group developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation4. The cationic lipid-like materials could encapsulate therapeutic mRNA in lipid nanoparticles by electrostatic interaction. To date, mRNA therapeutics is a promising strategy for disease treatment and vaccination. In contrast to DNA therapeutics, mRNA delivery results in transient expression of encoded proteins, and so avoids complications associated with insertional mutagenesis. Currently, mRNA therapeutics, including disease treatment and vaccination, are in the process of clinical trials. For instance, TranslateBio has conducted phase ½ clinical trials in delivering mRNA encoding fully functional cystic fibrosis transmembrane conductance regulator (CFTR) protein to treat cystic fibrosis by nebulization. For COVID-19, Moderna (co-founded by Dr. Langer) and Pfizer all utilize lipid nanoparticles to deliver mRNA encoding for a prefusion stabilized form of spike protein. Moderna also has two mRNA cancer vaccines in phase 1 and phase 2 to target solid tumors and melanoma. These clinical trials with mRNA delivery are incorporated to cationic lipid-like materials to enhance mRNA stability and lead to an increase in intracellular protein expression.

What engineers can do in biomedical field? 3

Fig. 3, Illustration for the formulation of lipid nanoparticles in mRNA delivery

In sum, biomedical engineering is a combination of multiple disciplines, such as engineering, biology, medical science, and chemistry. To date, biomedical engineers have contributed to the biomedical field in different aspects, such as new materials, fabrication methods, and medical devices to improve current medical treatments and solve emerged medical problems, for instance, COVID-19.

Reference:

1.        Guarecuco, R. et al. Immunogenicity of pulsatile-release PLGA microspheres for single-injection vaccination. Vaccine 36, 3161–3168 (2018).

2.        McHugh, K. J. et al. Fabrication of fillable microparticles and other complex 3D microstructures. Science (80-. ). 357, 1138 LP – 1142 (2017).

3.        Vegas, A. J. et al. Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nat. Biotechnol. 34, 345 (2016).

4.        Miao, L. et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat. Biotechnol. 37, 1174–1185 (2019).

Jason(Yen-Chun) Lu, All right reserved.