Gene therapy has been a hot topic in recent years, with the 2020 Nobel Prize in Chemistry being awarded for the CRISPR/Cas9 gene editing technique, and with skyrocketing global investment for research. In under a week in December 2020, the “big three” CRISPR companies — Crispr Therapeutics, Intellia Therapeutics, and Editas Medicine — received over $1.75 billion in new investment following the publication of new data that showed the successful treatment of just ten patients.
Besides CRISPR/Cas9, other gene editing techniques have been growing rapidly over the past decade, including Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Human Artificial Chromosomes (HACs). Each of these novel techniques, like CRISPR, has a wide range of strengths, weaknesses, and potential applications.
At Centaura, we work on developing HAC technology. Of the gene therapy techniques, we believe that HACs are the most versatile and powerful, capable of altering and inserting large arrays of synthetic genetic code. HACs not only treat genetic diseases, but can be used to customize the human genome to enhance human life on an unprecedented level.
Today, gene therapy is used and studied to transfer certain genes into human somatic cells, since changing the genome of germ line cells for therapeutic or other purposes is prohibited in most countries. Because of the nature of somatic cells, changes to genetic information using gene therapy are typically not inherited during division and only target certain cells in the body. This allows selective editing of certain body tissues and organ compartments like the eye or liver.
In this article, we’ll cover the basics of gene therapy, discuss how HACs work differently than other techniques, and show some of the countless clinical applications and advantages of HACs.
Gene therapy is a medical intervention that modifies the genetic material of living cells. It is a relatively young and rapidly developing treatment that was first used in 1973 to insert genes into E. coli cells, forming an intersection of genetic engineering and medicine. The first approved clinical trial for gene therapy in humans was in 1990, and the first gene therapy drug, treating a type of skin cancer, was approved in China in 2003.
Currently, more than two thousand gene therapies are at different stages of clinical trials. However, some clinical trials faced serious side effects, such as massive immune response or leukemia. Due to the variety of off-target effects and typically low efficacy of treatment, around 20 gene therapies have been approved for the actual treatment of patients so far in the United States.
For this reason, in order to harness gene therapy’s potential to treat more complex ailments, new gene therapy technologies must be invented.
Ex vivo & in vivo
There are two treatment approaches to gene therapy. Cells can be modified in vivo (directly inside the body) or ex vivo (outside of the body, for subsequent administration to patients).
The in vivo approach can target any cell of the body and is less invasive since it is performed through simple injection. This also makes it inherently less controllable and therefore less safe. It is challenging to limit the gene therapy modifications to only the cells of interest when the treatment is injected into the body.
In vivo gene therapy introduces genetic material directly by infusion. A solution containing a certain number of necessary genes, usually enclosed in carriers (or vectors), is injected into the body. The introduced genetic material reaches the target cells and is expressed there, coding for the desired protein products.
The ex vivo approach is more controllable and is a more common form of gene therapy. In ex vivo gene therapies, allgenetic manipulation is performed outside of the body. Cells are taken from the body, new genetic information is injected, and they are cultured and multiplied in number. After their genetic code has been modified, the cells are subject to a rigorous quality check to verify the proper changes before they are reintroduced to the body.
While ex vivo treatment is safer than in vivo treatment, only limited types of cells are possible to manipulate ex vivo. Brain cells can’t be treated or modified ex vivo, for example, since they cannot easily be removed or replaced. Ex vivo gene therapy is also a cell therapy, since it transplants genetically modified cells into the human body.
In most cases, viruses are used as carriers or vectors for gene delivery. Their natural ability to introduce genetic material into host cells can be not only harmful in the context of disease, but also useful in the context of gene therapy.
It is not as scary as it may sound: viruses are first lobotomized by genetic engineering methods, removing most of the genes responsible for virulence and rapid, uncontrollable spread. This frees up space for new genetic codes. Synthetic DNA can be loaded into the virus, which then delivers the new genetic material to cells.
However, the amount of free space inside viruses is limited. They are not designed for and not applicable to large human genes or arrays of genes, which leaves complex genetic conditions beyond the scope of the current gene or cell therapy approaches.
There has been astounding progress in the development of viral vector gene therapies, but these have hit a ceiling for the transmission size of the genetic load, and for the longevity of treated cells. There is a need for a vector to carry and express longer DNA sequences to treat more complex genetic conditions.
Artificial chromosome treatments
In order to provide longer-lasting, more complex, and personalized gene therapies, it will be necessary to invent gene therapy vectors that behave essentially like natural human chromosomes. The vectors should be stable in recipient cells, not rearranging or integrating into genomes, and they should express a large and complex set of genes and gene regulatory elements.
Is it possible to artificially synthesize a chromosome that acts and behaves like a natural one?
It is indeed. Scientists have already synthesized chromosomes for plants and mice, and have successfully added them to the genomes of living organisms. The organism treats the new DNA like its own, expressing it, replicating it, and passing it along during division.
The process to generate and express artificial chromosomes has now been demonstrated in several plant species by various laboratories. These include maize, rice, wheat, Brassica, barley and Arabidopsis. Entire biochemical pathways consisting of many genes could be combined on the chromosome, enhancing tolerance to various stresses such as drought or to various biological agents such as fungal, bacterial, viral, or insect pests. Among animals, artificial chromosomes have only been tested in mice, but not very successfully so far. Considering that mice are the most commonly used model animal, the scope of application for a mouse artificial chromosome is enormous.
Artificial chromosomes have shown potential to work with great effectiveness in other species. Once developed for humans, HACs will go beyond the limits of existing gene therapies to provide long-lasting, customizable, and selective genetic optimization.
HACs can be administered using the safer ex vivo treatment method to achieve a high level of precision and control. With ex vivo treatments, we can selectively modify only the cells needed for editing, avoiding dangerous genetic modification of non-target tissues. This specificity allows gene therapy with HACs to go beyond treating genetic diseases to enhancing human life altogether. Particularly in cases where new genes are being inserted to improve a healthy patient’s life, it is not worthwhile to use a less safe in vivo therapy.
While existing gene therapy techniques are limited by the capacity of viral vectors, HACs are not. Cells treated with HACs can be controlled for the expression of huge arrays of genes, and will pass their HACs along during replication, unlike viral vector treatments.
By inserting a new chromosome, treatments with HACs do not affect the existing genome, making them very safe. The inserted DNA can be controlled for expression using promoters, so any unwanted side effects can be easily removed. This is not an option for existing gene therapy techniques, where changes to DNA are irreversible and permanent, leading to horrible side effects if done improperly.
HACs are capable of delivering over 1,000 times more genetic information in a single treatment than other existing gene delivery vehicles. Rather than changing individual base pairs, one letter at a time, HACs can insert large sections of genetic code. By overcoming this technological barrier and successfully engineering HACs, we will be able to write entire new paragraphs into the story of our genomes, coding for a limitless variety of proteins to treat ailments and enhance human life.
Clinical applications of HACs
Due to HACs’ unique nature as a versatile gene editing tool to insert and express complex gene arrays, it has a huge range of clinical applications. HACs can be used to treat existing genetic diseases, or can be used to prevent and reverse ailments in the future.
Here’s a short list of some of the most accessible and attractive clinical applications of HACs:
Immune system rejuvenation
At Centaura, our first target for artificial chromosome treatments is immune system rejuvenation. Immune cells circulate with the blood throughout the whole body, letting them spread rejuvenating factors throughout all tissues and organs. Blood cell modifications need not be limited to rare genetic disorders.
If a patient receives our immune therapy at age 30 instead of 60, for example, we can prevent a multitude of diseases that an individual may be affected by later in life. We can rejuvenate and boost the immune system to prevent development of cancers and infections at later stages of life, for example.
The more genes we want to deliver, the larger carrier we need. HACs are able to deliver these large quantities of genetic material for immune system rejuvenation. Compared to existing gene therapies which are good for fixing rare mutations and defects, HACs are able to provide complex organisms with enhanced longevity and other desirable traits, such as a more robust immune system.
Blood cells can act as molecular fabrics and secrete therapeutic proteins directly into blood. In turn, this can have a positive systemic effect on many other organs and tissues.
GDF11 is a promising target. This circulating factor was first identified among the proteins that differ between the blood of young and old mice. In 2014, Science reported GDF11 as the first identified rejuvenating factor that can restore regenerative function. GDF11 possesses all the necessary characteristics: it is synthesized in the body, reverses aging in most tissues, and its level decreases with age. By forcing immune or other cell types to secrete this factor in blood we can see a positive effect on overall health.
It’s important to note that the above mentioned factors should be under strictly controlled concentrations. Otherwise, instead of beneficial effects, patients will develop negative consequences, as always happens when the dose is not controlled. In the case of rejuvenating factors, this could lead to the loss of cell function through rolling back to a less differentiated state of cell development. The risk of tumor transformation also exists. Genetic copy number control is extremely important for complex large genetic loads.
To keep the treatment under control, we should be able to switch production of these factors on and off depending on the individual’s health status. This is not currently possible with existing vectors, but as carriers, HACs will have the capacity to achieve this level of precision. A module can be inserted into the structure of HACs that can be used to toggle genes over the course of a lifetime. For example, genes could be turned on or off (regulated) using certain medications (pills). If dangerously high or low levels of factors are observed, they can be easily adjusted by toggling genes.
In certain degenerative diseases such as osteoarthritis, stem cells are depleted, and have reduced proliferative capacity and reduced ability to differentiate. Recently growing interest in regenerative medicine, using cell therapy and tissue engineering, where cellular components in combination with engineered scaffolds and bioactive materials were used to induce functional tissue regeneration.
Studies have shown that stem cell therapy can help reduce knee pain and improve function, representing a solution for curing joint diseases. But this has limited potential. HACs can be used to genetically modify stem cells ex vivo before transplant. This promises to have an even bigger effect because in addition to the desired stem cell effects, we can get health-improving effects by adding specific genes in HACs.
The HAS2 gene, responsible for synthesis of hyaluronic acid, can be incorporated into HACs. The patient’s isolated stem cells can be transfected with HACs and go through the selection process to enrich the population with the properly modified cells. Finally, these cells can be loaded into the joint, where they produce hyaluronic acid enzyme to start synthesis of hyaluronic acid. By being synthesized directly in the joint, this increases mobility and decreases pain.
In skin rejuvenation, the same enzyme can also be used as an alternative to short-term cosmetic injections which may result in inflammation.
One recent breakthrough in cancer treatment was the development of the Chimeric Antigen Receptor (CAR) T-cell therapy. This method modifies human T-cells ex vivo, making them express artificial receptors with an affinity for specific cancer cells. Thus, genetically modified T-cells become a weapon for selectively killing tumors.
Like CRISPR, CAR-T has been a hot field for new investment. In recent years, CAR-T IPO’s have been approaching $2 billion, financing rounds are around $4 billion, and market capitalization and trading of CAR-T companies has reached nearly $100 billion.
Nowadays, lentiviruses are commonly used for T-cell engineering but researchers are suffering from low efficiency of obtaining gene modified cells. Lentiviruses insert the genetic payload in randomly selected places of the genome that can potentially cause dangerous mutations and make it hard to select modified cells for further infusion. This greatly limits the scale of the technology and increases its price.
HACs would not have the same issues as lentiviruses. They promise a safer and a more efficient alternative by providing a selective vector for specific cells. Compared to CAR T-cell therapy, HACs can transfer larger loads of chimeric receptor genes in one place, and make cells synthesize them controllably, without any risk of causing mutations. The payload will stay apart from the cell genome and T-cell function will not be impaired. Cells modified with HACs can easily be selected ex vivo through the selection marker and only cells containing one HAC copy will proceed to further transplant.
Metabolic disorders develop during a person’s life. The most common is diabetes mellitus. Diabetes mellitus is characterized by high blood sugar levels that result from the body’s inability to produce and/or use insulin. Many people with Type 2 diabetes can manage their blood glucose levels with lifestyle changes and oral medication.
People with Type 1 diabetes can’t make insulin, so they must inject insulin to control their blood glucose levels. The injected insulin acts as a replacement for or supplement to your body’s insulin. Insulin pumps offer a solution: smallelectronic devices that mimic the way the human pancreas works by delivering small doses of short acting insulin in blood.
A more radical solution is to apply HACs to create an artificial pancreas consisting of genetically engineered islet cells to produce the hormones insulin and glucagon. Artificial pancreatic technology mimics the secretion of these hormones into the bloodstream in response to the body’s changing blood glucose levels. As a modification, implantable capsule for pancreatic islets has been designed to keep these cells inside and to produce insulin directly in bloodstream. This promises several years without insulin injection and without immunosuppressants for diabetes mellitus patients who require insulin.
Besides regenerating pancreas cells to treat diabetes, HACs can be used to create artificial tissues and regenerate other organs. For example, HACs can be used to regenerate the thymus in adults, reversing the deterioration that comes with aging, or to regenerate liver cells to treat hemophilia A.
Gene therapies have created an exciting field of medicine that is constantly evolving, and that still is emerging from its early years. Besides the CRISPR/Cas9 system, new technologies are being developed to maximize the power of artificially engineered DNA.
HACs are a particularly promising technology, which have huge potential to customize the human genome. HACs provide nearly infinite possibilities for disease treatment and life enhancement, and will be an exciting technology to arrive in the coming years.