Sickle Cell Disease (SCD) is an inherited disease that contains a mutation in a beta-globin gene. There are only a few potential treatments present these days, but even those treatments set limitation on a patient’s condition due to the high risk of side effects. A recent study has shown that SCD should deal with B-Cell Lymphoma/Leukemia 11A (BCL11A) genes which tell our body to start producing beta-globin genes that allow areas for SCD mutation. This contributed to the idea of treating SCD and setting goals that must be achieved such as 1) safe and efficient gene transfer, and 2) high-level and stable gene expression. For this inherited disease, high medical technologies, such as CRISPR-Cas9, ZFNs, or TALENs that are considered the best tools for targeting mutated site, have been used. The new strategies for treatment of SCD can be done by correcting mutated genes in red blood cells (RBC), inducing fetal hemoglobin (HbF) in a patient’s body, or adding certain genes together that can also enhance the production of HbF. In these processes, there are several challenges that block the application to humans; however, these challenges may be fixed by continuous efforts on improving strategies for better gene transfer and gene expression. The recent advances in genetic manipulation technology will bring treatments for a variety of inherited diseases including SCD in the near future.
Sickle Cell Disease (SCD) is an inherited red blood cell disorder, also considered as a point mutation (a genetic mutation where only a single nucleotide base is changed) in the beta-globin gene. People with SCD have abnormal hemoglobin called sickle hemoglobin, which is a malfunctioning version of hemoglobin that carries oxygen throughout the body. This occurs when abnormal hemoglobin forms strands that cause alterations in its shape. It then eventually drags the cells from flowing freely through the blood vessel and blocks blood flow in the vessel. The problem here is that sickle cells not only hinder the transferring of oxygen to the whole body, but also reduce the number of red blood cells. This is because sickle cells live up to only 20 days, a sixth of a normal cell’s lifespan, and people are not adapted to producing red blood cells fast enough to replace the sickle cells. This then leads to a shortage of red blood cells, a condition called sickle cell anemia, which causes people to lose energy.
Approximately 7% of babies are born with sickle cell traits and they suffer from their cells’ distorted shape that causes shortage in oxygen.1 Because this disorder can damage tissues and organs by disrupting normal delivery of oxygen, it can lead to life-threatening complications. Until now, there have only been a few potential treatments such as bone marrow transplants (also called stem cell transplant, which removes bone marrow stem cells of the patient using radiation and injecting healthy stem cells from a donor) or blood transfusions (adding extra blood which increases iron level in the body) that target SCD, but even those treatments set limitations on the patient’s age or condition because they are risky tasks that contain complicated process.2 In 2011, researchers successfully corrected SCD in mice, using one of the methods in gene therapy. However, gene therapy is yet to be used effectively in reality due to the high risks such as GVHD/graft (Graft Versus Host Disease) rejection with unrelated hematopoietic stem cell transplant (HSCT) donors.3 Due to this concern, researchers are continuously focusing on more effective and safer ways to perform gene therapy. Herein, new technologies and strategies targeting treatment of SCD will be discussed.
Target: Sickle Cell Disease
SCD is associated with a mutation caused by a single nucleotide substitution in the first exon of the beta-globin gene. This mutation causes hemoglobin to crystallize in a low-oxygen environment, causing red blood cells (RBC) to bend into a sickle shape. The sickle shape of the red blood cell blocks the vessel and leads to deoxygenation. RBC go through specific processes when a person physically develops. Gamma-globin genes are active during fetal hemoglobin (HbF, main oxygen transport protein) production, and beta-globin genes are active during adult hemoglobin (HbA) production. In newborns, HbF production is highly active in the vascular system because it has much greater attraction to oxygen than HbA and helps newborns to develop better during their fetal stage. After approximately 6 months, during the differentiation process, B-Cell Lymphoma/Leukemia 11A (BCL11A) genes activate beta-globin gene and cease the gamma-globin expression, which leads to the production of HbA.4 Here, when mutation occurs (thalassemia for example), this process is delayed for 3-5 years, which means that the production of HbF will continue for 3-5 years. Due to risks, such as age limitation, lack of matching donor, or allergic reaction, hematopoietic stem cell (HSC, self-renewing progenitor cells that differentiate blood cells) transplantation is the only way to cure SCD.5 In order for SCD to be completely treated by gene therapy, two main goals must be achieved: safe and efficient gene transfer, and high-level and stable gene expression.
Method: Gene Editing Technology
Just as medical technology has advanced at an unprecedented pace since the early 1990s, so has it tremendously contributed to the wellbeing and longevity of mankind. There are nevertheless countless diseases and medical conditions that have absconded from our meticulous work in biotechnology through genetic loopholes, and we have yet to research further to find the perfect cure in treating such diseases instead of temporarily containing them. Recently, however, the scientific world has perceived a glimmer of light on gene technology, such as TALENs, ZFNs, or CRISPR-Cas9, especially on gene therapy,6 a study that has given new hope to those who have potentially inherited diseases and conditions obtained from genetic malformation. Those gene editing tools are used to accurately insert or remove a specific part of a DNA sequence and have recently become a phenomenon throughout the world.
The CRISPR-Cas9 system stands for Clustered Regularly Interspaced Short Palindromic Repeats System. There are two primary molecules that manipulate the process: the cas9 enzyme and guide RNA (gRNA). The cas9 enzyme works as a pair of scissors that cut the two strands of DNA at a specific location so that the DNA sequence can be added or removed. The gRNA literally guides the cas9 enzyme to the specific location where the strand should be edited. The gRNA has about 20 RNA bases that are complementary to the DNA sequences. Due to this, the gRNA will only bind to the DNA where it may, which is the targeted part of the sequences (Figure 1. A).
The TALENs system stands for Transcription Activator-Like Effector Nucleases. In order to cut the specific location of the DNA, two FokI (Flavobacterium okeanokoites I, a restriction endonuclease that contains recognition sites for DNA-binding domain and non-specific DNA cleavage domain) enzymes must come together. One for each strand binds to two TALE (transcription activator-like effector) DNA-binding domains that are composed of 33 to 34 amino acid repeating strands to produce two TALENs. When two TALENs are met, FokI strands inactivate or provide space to insert a new gene. This system will enable the insertion of necessary genes (Figure 1. B).
The ZFNs system stands for Zinc-Finger Nucleases that offers the ability for target gene modification to specific genomic sites in cells. ZFNs are restriction enzymes created by fusing a zinc-finger DNA-binding domain, which contains 3-6 individual zinc finger repeats (>2 DNA binding domain of the protein) and 9-18 base pairs, to a DNA-cleavage domain, which consists of combination of N-terminal and C-terminal recognition domain. Among the DNA-binding domain, there can be many combinations with DNA bases; different combinations of zinc-finger proteins bind to different sequences of DNA. This fact not only increases the variability of DNA sequences, but also heightens the efficiency of binding to specific sites. Similar to TALENs, two FokI nucleases bind together to make a pair of ZFNs. This also enables a cut in the section of the DNA, which allows the injection of the needed genes (Figure 1. C).
Figure 1.(A) The structure and function of CRISPR-Cas9 system; (B) The structure and function of TALENs system; (B) The structure and function of ZFNs system.6
Results: Gene Therapy for SCD
SCD occurs due to the mutation in beta-globin genes. BCL11A notify gamma-globin genes, which produce fetal hemoglobin cells, to initiate the production of adult hemoglobin in beta-globin genes. There are many strategies to find treatments for SCD that are present these days, but it is hard to overcome the barriers. However, researchers continue to examine various ways to treat these inherited diseases as shown in Figure 2. The first approach is gene correction, which is substituting sickled beta-globin genes into normal beta-globin genes. The second approach is HbF induction. There are four ways to this approach. The first is to knockdown BCL11A, notifier of adult hemoglobin, using shRNA. By eliminating BCL11A, one’s body will only produce gamma-globin genes, which will lead to having a higher number of HbF compared to sickled hemoglobin. The second method is to disrupt the BCL11A enhancer. By blocking the expression of the enhancer, one’s body will not be able to produce BCL11A genes, which will lead to the same result as the first method. The third way is to force chromatin looping by putting in transcription factors. When several transcription factors are forcefully pushed into the chromatin, a loop that induces the HbF will form. The last approach is gene addition. By integrating lentiviral vectors (LV) that contain beta-globin, gamma globin, or antisickling beta-globin cassette, it can reduce the activation of surrounded gene expression, which will increase the chance of modifying mutated site, the beta-globin locus control region.7 Through this process, the number of HbF will increase, which in turn will reduce the relative number of sickled cells. Figure 2 depicts the overall strategy of various approaches for correcting the SCD using gene therapy.8
There are two approaches that are put into experiment for treating SCD. The first approach is to block the BCL11A expression since BCL11A plays a key role in producing SCD. If the expression of BCL11A is blocked, only the fetal hemoglobin (HbF) is produced. Then, the number of HbF will increase relatively to the number of sickle hemoglobin (HbS). The second approach is to treat SCD without interfering BCL11A. In this process, researchers inject a gamma-globin gene to increase the relative number of fetal hemoglobin cells. If the number of HbF outnumbers the HbS, since the lifespan of sickled-cells are much shorter than normal cells, the disease will gradually be treated. Between the two approaches, the treatment without interfering BCL11A is less efficient and riskier, so researchers are further researching on increasing the survival rate when treating SCD by interfering BCL11A. BCL11A is an important transcription factor required for B lymphocyte development. The researchers use lineage-specific micro RNA (shRNA) to slice the BCL11A gene. In this process, the engraftment of BCL11A on HSC (hematopoietic stem cells) increases, which means that beta-globin genes started their production. LCR (Locus Control Region) mediated on shRNA is efficient for BCL11A knockdown and gamma-globin induction. If the polymerase-dependent regulatory elements, which increase the expression of shRNA, are used, the engraftment of BCL11A on HSC gradually ceases, which leads to slicing BCL11A.Figure 3 shows knockdown of BCL11A reduces engraftment of HSC in both blood cells and the site of B cell production.7 Figure 3 (A) shows lentiviral vectors with the top vector that is manipulated with SFFV(spleen focus-forming virus)-shRNA vector for transplantation and the bottom vector as a control. In Figure 3 (A), the dark vertical black line represents the site where shRNA vector targets for the BLC11A. Figure 3 (B) shows clear difference in the number of gene-modified cells with and without the use of SFFV-shRNA in three different sites: B cells, monocytes, and granulocyte. After the treatment with SFFV-shRNA, significantly low amounts of BCL11A modified genes were present in all three sites, which proves the effect of the transplantation.
Figure 3. Knockdown of BCL11A reduces engraftment of HSC.(A) Lentiviral vectors: the dark vertical black line represents the site where shRNA vector targets for the BLC11A and the top vector is manipulated with SFFV-shRNA vector (for transplantation) and the bottom one is the control vector. (B) The number of gene-modified cells with and without the use of SFFV-shRNA in three different sites: B cells, monocytes, and granulocyte.
Figure 4. Efficient knockdown of BCL11A leads to high gamma-globin and HBF induction in human primary cells in vitro.(A) Quantification of Western blots derived from 3 independent experiments. Data shows the percentage of BCL11A expression in four different conditions. (B)Data shows the percentage of gamma-globin or HbF induction compared to control experiment.
In contrast to the controlled model, SFFV-shRNAmiR model shows significantly low rates of CD34+ cells (hematopoietic progenitor cells found in bone marrow and cord blood), meaning high rate of success in lentivirus-based transduction of cells. Successfully transduced cells showed a significantly low percentage of relative numbers of BCL11A expression (in both SFFV-shRNA and LCR-shRNA condition), which means that transduction of CD34+ cells will possibly reduce the expression of BCL11A genes which may lead to potential treatment of SCD. Figure 4 (A) shows the difference in BCL11A gene expression in control to SFFV-shRNA which is the result of success in transduction. Using these successfully transduced cells, Figure 4 (B) experiment shows that outstanding number of HbF was produced, which opened the possibility of having relatively higher number of HbF (fetal hemoglobin) than HbS (sickle hemoglobin).9 This experiment demonstrated another potential treatment for SCD.
Autologous Gene Therapy Using ZFNs
Currently, there is one way to treat Sickle Cell Disease (SCD), which is the allogeneic hematopoietic stem cell transplant, the injection of the normal stem cells coming from a donor to the patient.2 However, this treatment is not efficiently used due to the hardship in finding a fully matched donor. In order to attenuate this problem, researchers are examining autologous gene therapy, which is the way of using patients’ own hematopoietic stem cells (HSCs) and making them produce normal red blood cells. This has now become an ideal approach to gene therapy for SCD. Researchers tested this treatment and concluded that there can be a “gene correction”, which is the correction of the sickle mutation, and a “gene modification”, which is the change of a gene rather than the correction of the sickle mutation. In these treatments, ZFNs are utilized to target specific genomic sites.5 Nucleases create a double-strand break (DSB) where ZFNs are targeted. Then, two major DNA repair mechanisms correct those DSBs. The first mechanism is called non-homologous end joining (NHEJ). This mechanism can lead to the introduction of errors at the break site, making it not functional at all. The second mechanism is called homology-directed repair (HDR). In order for this mechanism to work, there should be a homologous donor template to repair the break. When the homologous donor template is no longer available, NHEJ can happen. However, inappropriate NHEJ can lead to the mutation of DNA, which can generate tumor cells.
SCD is an inherited disease and is caused by a mutation of a single nucleotide substitution in the beta-globin gene. This mutation leads to the change of hemoglobin crystallization, resulting in bending red blood cells (RBC) into a sickle shape. The sickle shape RBC delivers less oxygen to the cells in the human body than normal RBC, which initiates SCD. Gamma-globin genes are responsible for the production of fetal hemoglobin (HbF) in early age while beta-globin genes are active in later age to produce adult hemoglobin (HbA). Among the two types of hemoglobin, HbF has much greater attraction to oxygen than HbA. For the research of inherited diseases including SCD, recently, the innovative gene editing technologies such as TALENs, ZFNs, or CRISPR-Cas9 have been developed and investigated actively in the scientific world. Those gene editing tools make it possible to accurately insert or remove a specific part of a DNA sequence at a targeted site. Therefore, the gene therapy equipped with advanced gene technology potentially brings new treatments for incurable inherited diseases in the near future.
The goal for treating SCD is to prevent pain, infections, and organ damages. This goal can be achieved if the two objectives, safety and efficient gene transfer, and high-level and stable gene expression, are met.8 It is hard to have a high-level erythroid (immature red blood cell)-specific expression. In regulatory elements in the beta-globin locus control region (LCR), there are potent erythroid specific enhancers that enhance high-level erythroid expression. To activate more of the enhancers, it is necessary to find hypersensitive (HS) sites to find the areas where enhancers are present.10-12 Another challenge is that it is hard to recognize if the designed experiments has been processed successfully. In order to find out, the researchers should inject the insulators to the insulation site (mutation site) where they can block further expression of other endogenes. This will raise the efficiency of expression of the gamma-globin genes, which will eventually increase the amount of fetal hemoglobin.13 If these two goals are met, it is theoretically the success of new gene therapy for inherited diseases. However, practically, there are still some financial concerns dealing with profit or cost from developing a gene therapy. Also, many gene therapy approaches are individualized to each patient. It is nonetheless impossible to treat. In 2011, using the first approach mentioned previously, blocking the BCL11A gene, researchers successfully corrected SCD in mice.
The researchers used the CRISPR gene editing technology to correct the point mutated parts in SCD. These days, new technologies have been developed rapidly and researchers are continuously testing various strategies targeting SCD. Just like the three approaches, gene correction, HbF induction, and gene addition, there may be many more ways to advance the treatment for SCD. The overall challenge towards treating inherited diseases is that those diseases can only be potentially treated with gene therapy.
Treatment of inherited diseases is not limited by technology or strategies; rather, they are too risky in that they are yet to be readily treated because of many different characteristics that each individual has. Also, despite the efficacy of these technologies in treating experimental models of monogenic hereditary disorders, applying them successfully in a real human body is an unsolved problem. Hence, it is crucial to find the best approach that matches two objectives of treating inherited diseases and other individual factors. By the success in this new gene therapy, the world must put more focus on applying this system into human stem cells. However, since gene therapy deals with human genes that control basic instructions, it raises many ethical and social concerns.
SCD, an autosomal recessive disorder, can be treated by using gene therapy by gene insertion or editing in beta-globin gene production sites. Among the tens of millions of dedicated medical researchers, still a huge number of them make lifetime contributions to expand on the concept of gene therapy for SCD by putting their knowledge into meticulous practice, while the rest of us pray for their success and for the betterment of humanity. The world seems now focused much more than ever on the rapidly developing medical technology. The recent advances in genetic manipulation technology offer a new approach for treating a variety of inherited diseases including SCD.
I would like to thank Dr. Kaapjoo Park ([email protected]) for his excellent mentorship and guidance in the preparation of this paper.
About the Author
HyoJu Kweon, 17, USA
HyoJu Kweon is currently attending Maine School of Science and Mathematics (95 High St, Limestone, ME 04750, USA). She chose to transfer her school to science-related school to challenge her ability. She enjoys studying scientific topics and showed her confidence in the field of science by supporting Science National Honors Society that promotes interest in science to many people. Since now, she successfully achieved her academic goals marking high scores on GPA, AP, and SAT. She is looking forward studying and researching biology-related topics even after the college.
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