Biotech-2021, part 1

Overview of important Biotech news for the first half of 2021

Ilya Yasny, XX2 century

The pandemic in 2021 does not even think of ending, but not a word about it further: we will talk about the most interesting scientific developments related to health and medicines.

Photos of mitochondria in dynamics

Previously, scientists had one way to obtain high–quality images of cellular organelles - electron microscopy. In it, a stream of electrons is used to obtain images of objects. Any variation of this method requires fixation of the sample and leads to the death of the cell under study. Now scientists from the Royal Institute of Technology in Stockholm (KTH) a non-invasive method for obtaining three-dimensional images based on fluorescence has been developed. Fluorescence occurs when some substances are irradiated with ultraviolet light, while the molecules of the irradiated substance pass into an excited state with higher energy, and then emit light of a longer wavelength.

Usually fluorescent methods are used to obtain two-dimensional images. But the combination of several ingenious techniques made it possible for the first time to apply the fluorescent method to obtain dynamic 3D images, and even without destroying the cell. At the same time, a fast method of detecting large-area images, the interference phenomenon and a fluorescent dye with the ability to turn on and off were used. These tricks made it possible to build a high-resolution three-dimensional object (up to 50 nm) without interfering with the vital activity of the cell. Moreover, high-speed image capture with a frequency of 1-2 times per second made it possible to study live objects of such a small size in dynamics, which was previously generally unavailable with such a resolution. In general, it used to take 20 hours to record one such three-dimensional image.

The new method allowed researchers to study the localization and function of neural proteins, in particular, in synapses and axons, where organelles and proteins are so densely located and move so actively that it is impossible to study them with less rapid and sensitive methods.

So, scientists managed to obtain a three-dimensional image of mitochondria in a living cell. In the video, you can see a small mitochondria (green) and a larger neighboring one (red).

In Fig. 1 shows two images of the actin cytoskeleton at different points in time and a picture of the change between them (in blue). The enlarged picture below shows the spiral structure formed by the twisted strands of the cytoskeleton.

Fig. 1. Images obtained by a new method of fluorescence microscopy.

Perhaps in the future, similar methods will allow us to get closer to deciphering the secrets of the brain and better understand the mechanisms of pathogenesis of neurodegenerative, and many other diseases.

Excellent results in the treatment of cervical cancer

Cervical cancer is well treated at that stage until it has become metastatic (and even better, it is prevented with the help of a vaccine against the human papillomavirus). Nevertheless, despite vaccination and screening programs, 260 thousand people die from this disease every year. Newly identified patients receive chemotherapy, radiotherapy, and surgical treatment, but most of the tumors return. A few years ago, Keytruda (pembrolizumab), a drug belonging to the group of immune checkpoint inhibitors, was approved for the treatment of cervical cancer. However, the response to therapy was observed only in 17% of patients, and the median survival was 9.4 months, and then only in patients with high expression of the PD-L1 marker.

The success of the company's pembrolizumab antibodies Merck&Co and nivolumab companies Bristol Myers Squibb led to an explosion of developments aimed at inhibiting PD-1 and its ligand (binding protein) PD-L1. These proteins play the role of "brakes" of the immune system and are used by the tumor to avoid an immune response that could destroy it. The drugs that suppress them have taken an important place in the treatment of cancer tumors (and their sales in 2021 are estimated at $ 33 billion), and have allowed many people to be cured of cancer who would have inevitably died from it earlier. However, this is not yet a panacea: as with cervical cancer, responses to therapy rarely exceed 30% and scientists are constantly looking for ways to increase effectiveness.

The good news was the publication of the results of a comparative study of another such antibody Libtayo (cemiplimab). Sanofi and Regeneron. This is also an antibody against PD-1, and in a study in patients with metastatic cervical cancer, it showed a significant superiority over chemotherapy: median survival increased from 8.5 to 12 months, and the risk of death decreased by 31%. So far, this is the only drug that has shown such results in the third phase of a clinical trial, and even in monotherapy mode (that is, alone). So, Merck & Co published a press release stating that Keytruda also lengthens the life of patients, but only in combination with chemotherapy. An interesting question is why the properties of antibodies against the same proteins differ, but there is no definite answer to it yet.

Another promising approach for cervical cancer is cell therapy. While T-lymphocytes with a chimeric antigen receptor (CAR-T) have not yet managed to achieve good results in solid tumors (the so-called tumors that are not related to the circulatory system), unmodified lymphocytes isolated from the patient's tumor succeed. This method has been tried since 1985, but problems with the stability of cell product production did not allow achieving good results in the clinic. They were achieved only by Iovance, which showed good results in melanoma, lung cancer, head and neck cancer and, finally, cervical cancer. So far, this data is based on a modest number of patients (27), and there is no long–term data on survival, however, what there is is impressive: in 11 people, tumor growth stopped, in 9 the tumor partially decreased, and in 3 it disappeared completely! If we consider that many patients have already received chemotherapy and (or) anti-PD-1 antibodies (for example, Keytruda), then this result is very encouraging. It remains to wait for more mature data and make sure that everything is in order with production.

Fig. 2. Mechanism of action of tumor-infiltrating lymphocytes (TILs) of Iovance company. After administration to the patient, the cells migrate to the tumor, recognize the tumor antigens there (since they were isolated from the same patient, they are already capable of this). Then the destruction of tumor cells by activated T-lymphocytes occurs.

New malaria vaccine

Malaria is a terrible disease affecting mainly the population of Africa and Southeast Asia. More than 400,000 people die from it every year – which exceeds the death rate from covid – and there are still no highly effective vaccines and medicines. Attempts to create a vaccine against malaria have been made since at least 1910, and today there is one conditionally registered vaccine from GlaxoSmithKlein, Mosquirix. Its effectiveness is far from optimal: 56% a year after vaccination, and four years later the protection drops to 17% altogether. The most unpleasant thing is that a connection with an increased frequency of meningitis is not excluded for her. As a result, the vaccine has not yet been approved by WHO, additional studies are being conducted in African countries.

The development of Mosquirix began in 1984, based on the circum-sporozoic protein of malarial plasmodium (CSP) – one of the parasite proteins that is located on the surface of the plasmodium cell at the sporozoite stage when it enters a person (Fig. 3). CSP was combined with the hepatitis B virus protein HBsAg. The resulting chimeric protein forms virus-like particles and, when ingested, causes the formation of antibodies against CSP. The problem with Mosquirix is that the production process produces too much HBsAg protein compared to CSP, so the immune response to CSP is not optimal. Developers from the University of Oxford have achieved a vaccine in which there is no free HBsAg. In addition, the Matrix-M adjuvant of Novavax, the developer of one of the coronavirus vaccines, was used to increase the immunogenicity of the new vaccine (it was codenamed R21).

Fig. 3. The life cycle of malarial plasmodium. Both Mosquirix and R21 act at the sporozoite stage, when the parasite only gets from a mosquito into a person.

The new vaccine demonstrated 77% efficacy in a phase 2b study on 450 children aged 5-17 years in Burkina Faso. A year later, the study participants received a booster dose of the vaccine, which allowed them to raise the level of antibodies to the same as after the first vaccination. With Mosquirix, this could not be achieved. Interestingly, if a mosquito bites a vaccinated person, then antibodies get to him, and theoretically should reduce not only the incidence, but also affect the spread of infection.

To introduce the vaccine, research is needed in more countries, under more diverse conditions and with a longer follow-up period. However, so far we can talk about an unexpected success: R21 is the first vaccine that exceeded the WHO threshold for the effectiveness of antimalarial vaccines of 75%.

Mosquitoes help fight dengue fever

Another interesting way to combat tropical diseases is to use their carriers themselves, modified in a certain way. Such attempts are being made with regard to malaria, but so far quite a major success has been achieved in the fight against dengue. This disease is caused by the dengue virus, which is carried by mosquitoes, mainly of one tropical and subtropical species, Aedes aegypti. Every year, 50-100 million people get sick with the symptomatic form of dengue fever, and about 20,000 die, and tens of thousands more develop severe complications.

There are no specific antiviral drugs for dengue fever, so patients are treated symptomatically, and measures against mosquitoes are used as prevention: insecticides, nets, cleaning of reservoirs, but all these measures do not work too well.

There is a vaccine from dengue Dengvaxia company Sanofi, but it is not recommended for those who have not had dengue. Just as after an illness, after vaccination there is a danger of a more severe course of subsequent infection. One of the possible explanations is the phenomenon of antibody-dependent amplification of infection (which, by the way, everyone was so afraid of for SARS-CoV-2, but in reality it has never been observed). And the effectiveness of Dengvaxia is not the most outstanding: 60% against symptomatic disease, 80% against severe form.

In June 2021 in The New England Journal of Medicine published the results of a different approach: mosquitoes were infected with their own intracellular parasite, the Wolbachia bacterium, which makes the mosquito resistant to infection with the dengue virus. Such mosquitoes should be released into the wild regularly for several months, and then nature does its own thing: wolbachia allows only infected mosquito offspring to survive.

Fig. 4. How does wolbachia help against the spread of dengue? The red mosquito is infected with wolbachia, but the green one is not. The virus does not replicate inside a wolbachia-infected mosquito, so the mosquito does not spread the virus when bitten, unlike an uninfected one. Wolbachia probably affects the mosquito's immune system, and also competes with viruses for limited cellular resources.

In order to test the effectiveness of this measure, a cluster randomized clinical trial was organized in one of the provinces of Indonesia: infected mosquitoes were released in 12 districts, and 12 others were left as controls. Then the study recruited volunteers who were admitted to the hospital with acute fever, and they were tested for dengue. 2905 participants were recruited in areas with infected mosquitoes and 3401 in control areas.

It turned out that the new measure protects against the disease by 77%, and from hospitalization – by 86%, that is, better than a vaccine! There is some danger that the virus will develop resistance to wolbachia, but so far no such cases have been observed.

There are other strains of wolbachia, with the help of which it may be possible to cope with Zika, Chikungunya, yellow fever and Mayaro viruses, which are carried by the same mosquito. At the moment, 7 million people live under the protection of Wolbachia, and the goal of the World Mosquito Program is 75 million people by 2025 and more than half a billion by 2030.

Chimera of man and monkey

A team of scientists from the USA, China and Spain created embryos consisting partly of human, partly of monkey cells, and they lasted 20 days. It sounds a little creepy, but such experiments are needed for at least three reasons:

• study of embryonic development,
• development of new therapies,
• growing organs for transplantation.

Such manipulations with human embryos are prohibited, so scientists circumvent this prohibition by using chimeric organisms. It is not a fact, however, that there will not be difficult ethical problems on this path either.

But this experiment is the very beginning of such studies, and there is nothing to be afraid of yet: the embryos were not implanted anywhere and by the 20th day of cultivation in laboratory conditions they died themselves. In addition, the scientists consulted with independent experts in the field of bioethics before carrying out the work in order to ensure the most ethical conduct of the experiment.

Each of 132 six-day-old macaque embryos was injected with 25 pluripotent human stem cells. Potentially, such cells can turn into any cell, not only of the body, but also of the placenta. Previously, scientists carried out such manipulation with mouse and pig embryos, but human cells did not take root – these species are too evolutionarily far from humans. For the first time, the experiment was successful with macaques.

Fig. 5. The scheme of the experiment on implantation of human cells to the monkey embryo. d.p.f. – days after fertilization. IF – immunofluorescence analysis, scRNA-seq – unicellular RNA sequencing.

To do this, we took pluripotent cells, additionally reprogrammed by genetic engineering methods to give them more stem potential (the ability to differentiate into cells of different types). Human cells were marked with a red protein marker so that their fate could be easily monitored. On average, after 15 days after implantation, 3-4% of the embryo's cells turned out to be human, in one embryo this number reached 7%.

It has been shown that human cells in general do not impair the development of the embryo compared to control samples, but prefer to develop not into all types of cells, but only into some. So, they could not turn into trophoblasts – cells of the outer shell of the blastocyst. Single-cell transcriptomic analysis He showed that monkey cells are not the same as in control embryos – further experiments revealed that they were influenced by the signals they exchanged with human cells. In the future, it will be interesting to see exactly how these signals affect cell development.

It is also necessary to study what will happen if cells of other lines are introduced, a different number of them at other stages of embryo development.

Optogenetics: the first success in restoring human vision

Optogenetics is a complex of biomedical methods based on the genetic engineering of neurons, forcing them to express photosensitive receptors in order to further influence them with light with a certain wavelength, prompting certain reactions. One of the promising applications of optogenetics – the return of vision to the blind – has been developing since the 2000s, and finally, the first success has been achieved in humans: a patient with retinitis pigmentosa, who previously could only distinguish light from darkness, was able to find objects on the table with special glasses a year and a half after treatment.

With retinitis pigmentosa, due to a genetic defect, photosensitive retinal cells – rods and cones – gradually degrade, which causes a person to gradually go blind. While it is impossible to cure this disease, it is only possible to delay the loss of vision with the help of vitamin A and other means. They are trying to restore vision with the help of retinal transplantation, stem cells, or to compensate for the defect with gene therapy methods. However, so far all these techniques have not led to decisive success. Perhaps optogenetics will succeed in the end.

In this method, scientists deliver the gene encoding the photosensitive protein not to retinal cells (which the blind have almost no left), but to nerve cells that carry the signal further to the brain (ganglion cells), thus turning them into photoreceptor cells. Interestingly, the photosensitive protein is not human, it is borrowed from the green algae chlamydomonas. This is the membrane protein ChrimsonR, which, when irradiated with light at a wavelength of 590 nm, opens a channel in the cell membrane. Through this channel, positively charged ions enter the cell, which causes its excitation and further conduction of the nerve impulse.

The patient is given one injection of an adeno-associated vector into the eyeball (purple in Fig. 6), carrying the desired gene. Then, after the protein expression reached the desired level (it takes several months), the patient began training with special glasses – in fact, it is a digital camera that converts external signals into monochromatic light at the wavelength that best excites the protein. At first, the patient's brain did not know how to interpret such unusual signals. However, gradually, as the training progressed, the patient correctly learned to distinguish objects on the table in 92% of cases.

The achieved level of vision restoration does not yet allow to distinguish faces or read, but the beginning is a big trouble: 15 years have passed since vision was returned to mice using this method, so further improvements are not far off.

Fig. 6. Schematic representation of the principle of operation of optogenetic vision therapy.

Gene therapy of cardiovascular diseases

Now gene therapy is used for a limited number of rare diseases, it is a very expensive and piece-by-piece product. However, few doubt that in the future, with the development of technology, gene therapy will become more widespread and accessible. The only question is in which area and when it will happen for the first time. This half-year, two companies have announced the success of developments in the field of cardiovascular diseases, however, so far only in monkeys. But this is also a big step forward: previously, such results were obtained only in mice, and they are too far from humans to give confidence in subsequent success.

We are talking about the inactivation of the PCSK9 gene, which encodes a protein responsible for an increased level of low-density lipoproteins (LDL) in combination with cholesterol (the so-called "bad cholesterol"). In people with an inactivating mutation in this gene, the frequency of many cardiovascular diseases is significantly reduced. There are already antibodies against this protein on the market, but they require constant injections, because they are excreted from the body within a few weeks. It's quite another thing to create a gene therapy that will turn off this gene once and for all.

And so, in the work of the team from the company Verve Therapeutics presents the results of gene editing in macaques, which allowed to reduce the expression of PCSK9 in the liver by 90% and achieve a reduction of cholesterol in the blood by 60% for as long as 8 months (at the time of publication – and this is not the end of the experiment). At the same time, there were no adverse events in the form of hepatic toxicity, nor an immune response to the injected drug. The great advantage of the drug is that it mainly enters the liver, where it is necessary to turn off the expression of PCSK9.

The BioNTech and Moderna coronavirus vaccines are similar to the drug itself: mRNA encoding the DNA editing enzyme and the guide RNA indicating which part of the DNA to edit is packed into liposomes (roughly speaking, fat bubbles from precisely selected lipids). The scientists did not use the classical CRISPR-Cas9 gene editing system, but a more accurate (but also less flexible) base editing system that converts adenine (A) into guanine (G) at a certain position. This system does not contribute to DNA of double breaks, fraught with a large number of errors. Indeed, genomic analysis of monkey cells showed that inappropriate editing was extremely rare.

Fig. 7. The scheme of editing the bases. The guide RNA binds complementarily to the DNA at the editing site. The enzyme (purple) converts adenine (A) into inosine (I), which should not be in DNA. It also cuts the opposite strand of DNA, the cell's repair mechanisms complete it, and DNA polymerase inserts cytosine (C) opposite I on the principle of complementarity. Then the repair mechanism replaces I with guanine (G), because there is C in the opposite chain.

So far, this is the result on four monkeys, and there are no guarantees that people will be able to reproduce it, but the prerequisites are very good.

It is encouraging that another group of scientists, from Precision Biosciences, was able to achieve a safe reduction in PCSK9 expression by 85% and LDL level by 56%, and already on six macaques and within three years. A slightly different approach was used here: cutting is carried out using the enzyme meganuclease from chlamydomonas, and its gene is delivered to cells by the adeno-associated vector AAV8.

Another news from the field of gene editing: CRISPR-Cas9 was first used in adults in vivo, that is, the editing took place directly in the body. In June 2021, the first results of the use of gene editing directly in humans were published. The company Intellia (founded by 2020 Nobel Laureate Jennifer Dudnaya) and Regeneron reported data from the first six patients with ATTR-amyloidosis (a rare disease in which mutations in the TTR protein lead to its deposition in the heart, nerve fibers and other organs) 28 days after administration of the drug. All patients had a decrease in TTR in the blood, and in the group with a higher dosage – by an average of 87%. This result is close to what Alnylam's patisiran drug showed, but there is hope for one injection for life instead of injections every three weeks.

The drug, as in the case of Verve, is a liposome with two RNAs: one encodes a Cas9 protein capable of cutting DNA, the second is a guide RNA (gRNA). In Fig. 8 shows the scheme of the drug.

Fig. 8. Scheme of operation of the drug NTLA-2001.

The most remarkable thing is that after the company shows long–term effectiveness and safety in one liver disease, it will be very easy for it to switch to others: it only needs to change the sequence of the guiding DNA. Of course, there is no certainty that this approach will work for any diseases, but the potential is very great.

Google Dermatologist

Google has announced the launch of an application that will help determine whether there is cause for concern and whether to consult a doctor if a person finds something suspicious on his skin. The user should take a picture of the place that bothers him from three angles, answer a few questions about himself and his skin, and then a machine learning algorithm will assume that it can be based on a list of 288 possible pathologies.

According to Google, the search engine annually processes ten billion requests about skin problems, but words are often difficult to express what a person sees on his skin. Therefore, three years ago they started developing this algorithm, and in 2020 they published an article in the journal Nature. It was shown that the algorithm is not inferior to dermatologists in terms of the accuracy of the diagnosis and surpasses conventional therapists. And in another work it was shown that the accuracy of diagnoses made by therapists using this AI algorithm approaches the accuracy of diagnoses of professional dermatologists. Now the algorithm uses information obtained from 65,000 images annotated by dermatologists.

However, while the application is not planned to be used as a replacement for a dermatologist's examination and, if necessary, a biopsy: to do this, prospective clinical studies must first be conducted. So far, the company's algorithm has received a CE mark in Europe as a Class I medical device. These are devices of the least risk, such as glasses and thermometers, not intended for making medical decisions.

Fig. 9. The appearance of the Google Derm Assist application.

Currently, the application is available only to beta testers, but Google plans to expand its user base in the near future.

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