Following the completion of the Human Genome project in 2003, a notable fall in the cost of sequencing and therefore a much larger database of sequenced DNA from disease sufferers, research into genetics now allows scientists to develop exciting new treatments for many otherwise untreated medical diseases. At the forefront of this research involves the therapeutic possibilities of genome editing, which involves making targeted changes to the genome of diseased individuals by either modifying DNA sequences to alter certain characteristics, removing specific disease-causing regions of a genome, or adding transgenes (genes from a different organism) to specific locations on a genome. For the purpose of this essay, I will solely be focusing on therapeutic genome editing techniques, rather than venturing into the preventative procedures that often raise controversial ethical debates, such as the engineering of a genome before birth or conception and the risks that accompany this. Following a brief introduction into the basic science of each consecutive method of genome editing, I will be exploring the possible therapeutic use of genome editing to treat Hemophilia B as well as Sickle Cell Anemia, before delving into the use of disease-induced monkeys in order to study the causes and mechanisms of various neurological disorders.
Genome editing involves programming nucleases to “break” a section of DNA by creating double-stranded breaks at specific genomic loci before inserting a new DNA sequence by harnessing the natural repair mechanisms of cells. Rapidly advancing technologies have radicalized this field of study, bringing genome editing to the forefront of medical research. There have thus far been four types of programmable nucleases in order to modify a genome: meganucleases, zinc finger nucleases (ZFNs), TALENs and finally CRISPR/Cas9.Meganucleases are known as “molecular DNA scissors” and require protein engineering in order to change the DNA recognition sequence. ZFNs, designed in 1996 at John Hopkins University, are proteins with one end that recognizes a specific DNA sequence while the other end “cuts” the sequence. While they were the first programmable genome-editing tool, their disadvantage lies in the need for the researcher to engineer a new protein for each specific sequence, which is time consuming and often unsuccessful. Transcription activator-like effectors (TALENs) are proteins produced by Xanthomonas bacteria (sciencemediacentre.org/genome-editing) that can be easily programmed to recognize different DNA sequences and are thus more efficient and effective than ZFNs. On the other hand, TALENs are much larger than ZFNs and are thus harder to insert and maneuver within cells. Finally, the most up-to-date technology uses CRIPSR (clustered regularly interspaced short palindromic repeats) which are palindromic DNA sequences found naturally in the bacterial defense system, in between which the bacteria inserts a section of the viral DNA and produces complementary RNA that can thus recognize and destroy the viral DNA in the event of a second attack. Scientists can artificially synthesize CRISPR extremely quickly and at low cost. This technology works for non-monogenic diseases (diabetes, heart disease, neurological diseases) since the RNA-guided proteins can be re-targeted to a new sequence by altering a section of an accompanying RNA guide. Once the double stranded breaks are formed, depending on the cell state and the presence of a repair template, the sequence is reformed by one of two methods. NHEJ-mediated repair rejoins the two ends of the DSB without the presence of a repair template; however, this can lead to a nonsense mutation and thus the production of nonfunctional truncated proteins. Another process is called HDR, and uses an exogenous DNA template to rejoin the sequence and can thus replace a faulty gene and introduce a wild-type allele.
One specific monogenic genetic disease which could be treated by various genome editing techniques is Hemophilia B, a sex-linked recessive genetic disorder caused by a missing or defective clotting factor IX leading to increased bleeding and clotting time. Hemophilia B is typically treated with frequent clotting protein infusions, which is not only costly but could stimulate the immune system to mount a response and synthesize antibodies which prevent successful treatment. Prior gene therapy techniques also had various disadvantages, such as their inability to hone in on a specific DNA sequence, meaning regulatory genes may create off-target cuts that can actually triggers an alteration, such as leukemia. In 2011 Katherine High of the Children’s Hospital of Philadelphia successfully designed ZFNs for the factor 9 gene (F9) that were able to restore normal gene function to mice induced with hemophilia B. The researchers used two versions of an AAV (adeno-associated virus) that had been modified, with one carrying ZFNs to splice the DNA at a specific gene locus while the other carried the correct factor 9 gene. While previous studies had successfully performed ex vivo genome editing, this would often not be feasible for many human diseases which affect an entire organ system. Thus, the success of this study in which the mice that received ZFN treatment produced enough clotting factor to reduce blood clotting to regular levels without any side effects indicated an exciting prospect for a similar treatment of humans suffering from this disease. In fact, hemophilia B may be one of the most successfully treated genetic diseases in the future, due to the fact that it only requires the recovery of over 1% of factor IX activity in order to restore the blood clotting cascade and prevent serious blood loss. This is significant when analysising the efficiency of possible genome editing therapies, by analyzing the fitness advantage that the edited cells have over the mutated cells, such as whether they will grow larger or more powerful than their mutated counterparts. Hemophilia B is an example of a disease that does not require a significant fitness advantage to be able to achieve normal gene physiological function, since even a small change in the gene product levels can influence the clinical outcomes.
Furthermore, there are numerous research operations concentrating on the treatment through therapeutic genome editing of sickle cell anemia. Gang Bao at the Georgia Institute of Technology is currently using CRISPR to correct the defective gene of hematopoietic stem cells which had been removed from the bone marrow of a patient, before returning the corrected genes back into the patient, an example of a possible ex vivo treatment of the disease. A particularly fascinating alternative method of treatment involves harnessing the use of mutations in non-coding regions of the genome which provide resistance against specific diseases, by introducing these mutations by NHEJ into the patients to reverse the effects of the disease. In relation to sickle cell anemia, it has been proven that one factor that directly correlates to the severity of the disease is the degree of fetal hemoglobin in the blood. One version of the BCL11A gene which releases a product that decreases the HbF levels in the blood, is currently being trialed for possible therapeutic treatment.
Genome editing is now being used to model complex neurological diseases such as Schizophrenia, Alzheimer’s, Parkinson’s and autism in primates to study the mechanisms that allow the disease to progress. The use of rodents to model brain disorder, although extensively researched, has thus far been deemed futile due to the many differences in neurological pathways between humans and rodents and because the social behaviours are too different to be able to correctly study. Guoping Feng of MIT and Feng Zhang of Broad Institute and McGovern Brain Institute plan to alter the SHANK3 gene in embryos of macaque monkeys in order to induce autism and study the neurological science of the disorder and test possible drug treatments. Since neurological disorders are often polygenic diseases, CRISPR genome editing allows researchers to systematically test a large array of genes to determine if a mutation or a combination of mutations directly cause a disease or are simply indirectly associated to the disease.
To conclude, while genome editing has not yet been officially introduced into clinical treatment procedures, it opens up an exciting new field of study that could provide radical new treatments for diseases that have been otherwise untreatable by current medical practices. However, there are numerous improvements that need to be made to increase the effectiveness of genome editing: increasing the number of cells that undergo modification by improving the efficiency of HDR-mediated repair (since NHEF is already rather efficient since it is a natural process in most cell types), and improving the specificity of the technology to reduce the number of off-target mutations that can lead to serious side effects by making more use of CRISPR or Cas9 technology which is far more advanced and precise. Overall, genome editing is a rapidly advancing technique which could one day completely revolutionize the field of medicine, from introducing embryonic genetic modification to one day eradicating many of the diseases that are so destructive in our current society.
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