Review Paper: Tackling β-Thalassemia through CRISPR

13 min readApr 10, 2023

The future of combatting blood diseases via the smallest unit of the human genome

An animated image representing both red and white blood cells present in the blood stream.

Abstract:

Through recent studies, advancements in molecular biology and genetics have greatly extended our knowledge of hematological diseases. With such advances, the use of the CRISPR system has been interlinked in order to treat the debilitating disease of β-thalassemia.

β-thalassemia is a genetic disorder caused by mutations in the HBB gene, which encodes the beta-globin subunit of hemoglobin. Current treatments are highly associated with risks, though, due to the genetic nature of this condition, the use of the CRISPR system is an ideal tool.

This document hold the purpose of featuring a detailed report of the the use of CRISPR to treat beta-thalassemia. This technique shows great potential and holds tremendous promise as a potential cure for this debilitating disease. Topics will be discussed as followed by the headers down below.

Paper outline:

1.0: CRISPR in a Nutshell

2.0: Blood Disorders (general)

3.0: β-thalassaemia

3.1: Causes

3.2: Symptoms

3.3: Diagnosis

3.4: Current Solutions

3.5: Shortcomings of Current Solutions

4.0 Why CRISPR?

4.1: β-thalassaemia CRISPR Treatment

5.0: Clinical Trials

5.1: CTX001

5.2: EDIT-BTH

5.3: LUMINATE

6.0: Conclusion

1.0 CRISPR in a nutshell

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a tool that allows scientists to precisely edit DNA. It is a technology that has revolutionized genetic research and permits the removal, alternation or addition of genes in a very precise manner.

The CRISPR system is derived from a natural defense mechanism that bacteria use to defend themselves against viruses. The CRISPR system consists of two main components: the guide RNA (gRNA) and the Cas enzyme.The gRNA is a short piece of RNA that is designed to match a specific DNA sequence. The Cas enzyme is a type of protein that can cut DNA. When the gRNA and Cas enzyme are combined, they form a complex that can find and cut the specific DNA sequence targeted by the gRNA.

By introducing a specific gRNA into cells, scientists can direct the Cas enzyme to cut the DNA at a specific location. This creates a break in the DNA strand, which can be repaired by the cell’s own DNA repair machinery. By controlling the repair process, scientists can introduce specific changes to the DNA sequence.

CRISPR has many potential applications, from correcting genetic mutations that cause disease to creating new crops with desirable traits. The technology is relatively simple and inexpensive to use, which has made it widely accessible to researchers around the world. Additionally, this system can be highly specialized allowing it to be suitable for a wide variety of applications.

2.0 Blood Disorders (general)

Blood disorders are conditions that affect the components of the blood, such as red blood cells, white blood cells, and platelets. These components play important roles in various bodily functions, including oxygen transport, immune response, and blood clotting. When there is a problem with any of these components, it can lead to a blood disorder. They are generally classified as conditions which affect the generative process of blood, the transportation requirements of blood and the contents of the blood itself (oxygen, platelets, plasma).

Blood disorders can be broadly classified into three categories based on their underlying causes. The most common includes hematologic disorders are disorders that affect the production, function, and lifespan of blood cells. Hemostatic disorders affect specifically the ability of blood to clot, usually being fatal. Vascular disorders are the last type in which the blood vessels can excessively bleed and clot.

Furthermore, they can also be classified based on the type of blood cell they affect. Common ones include red blood cell disorders which affect the production, function, or lifespan of red blood cells, white blood cell disorders which affect the production, function, or lifespan of white blood cells and platelet disorders which affect the production or function of platelets.

It is important to note that many blood disorders can overlap in their symptoms and many times posses underlying causes.

3.0 β-thalassaemia

β-thalassemia is a genetic blood disorder that affects the production of hemoglobin, a protein found in red blood cells that is responsible for carrying oxygen throughout the body. People with β-thalassemia have mutations in the HBB gene, which provides the blueprint for making the β-globin sub unit of hemoglobin. These mutations can result in reduced or absent production of β-globin, leading to a shortage of functional hemoglobin in the blood.

There are two main types of β-thalassemia: β-thalassemia major and β-thalassemia minor. β-thalassemia major, also known as Cooley’s anemia, is a severe form of the condition that can cause life-threatening anemia, jaundice, enlarged spleen and liver, and bone deformities.

3.1 Causes

β-thalassemia is most commonly found in people of Mediterranean, Middle Eastern, and Southeast Asian descent, but it can occur in people of any ethnic background. People from these regions may be carriers of the HBB gene mutation even if they do not have the condition themselves, due to the high prevalence of the mutation in these populations.

The HBB gene mutations that cause β-thalassemia are inherited in an autosomal recessive pattern, which means that a person must inherit two copies of the mutated gene, one from each parent, to develop the condition. People who inherit one copy of the mutated gene are carriers of the condition but do not usually have any symptoms.

The risk of having a child with β-thalassemia is increased if both parents are carriers of the condition. In some cases, β-thalassemia may occur spontaneously due to a new mutation in the HBB gene, without any family history of the condition though this is rare.

3.2 Symptoms

Generally, the symptoms of β-thalassemia can vary depending on the type and severity of the condition, β-thalassemia major being more fatal.

β-thalassemia major presents symptoms such as severe anemia, pale/jaundiced skin and eyes, an enlarged spleen and liver, delayed cognitive and physical development and problems in systems such as the endocrine, cardiovascular and skeletal system.

β-thalassemia minor on the other hand shows symptoms which many times go unaddressed as they overlap with the common iron deficiency. Some include pale, cold hand and feet, weakness/fatigue, shortness of breath and dizziness.

Additionally, individuals with β-thalassemia may also experience high infection rates and complications leading to organ failure.

3.3 Diagnosis

The diagnosis of β-thalassemia involves a combination of physical examinations, blood tests, and genetic testing. Testing can occur when an individual suspects that they may have β-thalassemia or individuals who have the HBB gene can go through pre-natal testing.

Physical examination are the most common sign of β-thalassemia. Signs of anemia, such as pale skin, fatigue, and shortness of breath can be symptoms of β-thalassemia as they many times overlap with iron deficiency. They may also check for an enlarged spleen or liver, which can be a prevalent indicator of β-thalassemia.

Blood tests are another way β-thalassemia can be tested for. Through a complete blood count (CBC) test, the levels of hemoglobin, red blood cells, and other blood components will be measured. People with β-thalassemia typically have low hemoglobin levels and small red blood cells. A hemoglobin electrophoresis test can also be performed which separates different types of hemoglobin to identify abnormal patterns.

If a blood tests suggest the possibility of β-thalassemia, genetic testing is required to confirm the diagnosis. This involves analyzing a sample of the person’s DNA to identify mutations in the HBB gene.

It is important to diagnose β-thalassemia early to start appropriate treatment and prevent complications as this condition is deliberating and if left untreated, can many times be fatal.

3.4 Current solutions

All current solutions to β-thalassemia do not prove to have full term cures are many issues come up such as inaccsesibility.

The most common treatment is blood transfusions. Regular blood transfusions can help to increase the hemoglobin levels in people with β-thalassemia major, who have severe anemia. Another treatment plan is iron chelation therapy used to remove excess iron from the body, which can accumulate due to frequent blood transfusions. This therapy involves medication that binds to excess iron in the bloodstream and removes it from the body.

Bone marrow transplants are procedures in which a person’s bone marrow, which produces blood cells, is replaced with healthy bone marrow from a compatible donor. This is by far the most effective procedure as it has a moderately high chance of potentially curing β-thalassemia.

Similarily to any other deficiency, supplementation of folic acid is many times administered. Follic acid a type of vitamin B that is necessary for red blood cell production, a treatment is usually used as a short-term aid.

Specialized antibody-rich vaccinations are also administered. People with β-thalassemia have an increased risk of infections, so vaccinations against certain infections such as influenza and pneumococcal disease can be lifesaving.

Hydroxyurea is another medication that includes properties which stimulate the production of fetal hemoglobin, a type of hemoglobin that is normally present in newborns and can help to compensate for the defective hemoglobin in people with β-thalassemia.

3.5 Shortcomings of Current Solutions

The current standard of care for β-thalassemia are as mentionned as above and enclose dangerous shortcomings.

Blood transfusions, the most common treatment, frequently lead to serious conditions such as iron overloads. Frequent blood transfusions path towards the accumulation of iron in the body, which can cause organ damage over time.

Chelation therapy can help remove this excess iron, but it can be time-consuming and may have side effects. Many times, this therapy is too tenacious and the patient’s body is too weak to sustain it.

Inaccesability is also a huge concern due to the large dependence on blood donors. Due to patients with β-thalassemia requiringregular blood transfusions, they rely on a frequent supply of blood from donors. Availability of compatible blood β-thalassemia is also a concern as patients may require blood that is matched for specific antigens. Finding compatible blood can be challenging, particularly for patients from certain ethnic or racial groups. This can be life-threathing in areas with limited resources or during times of emergency.

The heightened risk of infections is also prominent for individuals with β-thalassemia. Blood transfusions carry a risk of infection, which can be particularly dangerous for patients, who usually have weakened immune systems as a result of their condition.

Other factors such as the cost of frequent blood transfusions and chelation therapy can be high, and may be a barrier to access for some patients. Furthermore, the limited effectiveness of the current solutions cause large problems. While blood transfusions and chelation therapy can manage the symptoms of β-thalassemia, they do not cure the underlying genetic condition. As a result, patients may require lifelong treatment. Due to such, the possible psychological impact must also be considered. Living with a chronic condition like β-thalassemia can take a toll on a patient’s mental health, leading to anxiety, depression, and social isolation.

4.0 Why Use CRISPR?

CRISPR, as previously mentionned, is a gene editing tool that can very precisely target and modify DNA sequences in living cells. With the potential CRISPR posseses, is seems very plausible to work towards correcting the genetic mutations that cause β-thalassemia.

There are several reasons why CRISPR may be an attractive option for treating β-thalassemia, the first being the high level of precision. CRISPR allows for precise targeting of specific genes, which means that it can potentially correct the genetic mutations that cause β-thalassemia without affecting other genes. Additionally, unlike current treatments for β-thalassemia, which only manage symptoms, CRISPR has the potential to provide a cure by correcting the underlying genetic mutations. This would mean patients could make a full recovery and hereafter, will not need any alterior treatment.

Another plus by using CRISPR is its cost-effective nature. While the development of CRISPR-based therapies is still in its early stages, it has the potential to be a cost-effective treatment option for β-thalassemia in the long run, especially if it can provide a cure.

4.1 β-thalassaemia CRISPR Treatment

As discussed, CRISPR-Cas9 is a genome engineering technique that offers novel possibilities for treating β-thalassemia.

There are several strategies being explored to use CRISPR to treat β-thalassemia, which is caused by mutations in the HBB gene that codes for the β-globin subunit of hemoglobin. One approach is to use CRISPR to correct the mutations in the patient’s own cells, while another approach is to create healthy cells in the lab and then transplant them into the patient.

In the first approach, CRISPR is used to introduce precise changes to the patient’s own DNA, which can potentially correct the mutations that cause β-thalassemia. This is done by designing guide RNAs that target the specific mutations in the HBB gene, and then using the CRISPR protein to cut the DNA at those sites. The cell’s natural DNA repair mechanisms then come into play, and the mutations are corrected. This approach is challenging, given the variety of mutations that can cause β-thalassemia, and is limited to the correction of known mutations of the HBB gene

Though this approach has shown promising results in preclinical studies, there are still significant challenges to overcome before it can be used in humans. One major concern is the risk of off-target effects, where CRISPR makes unintended changes to other parts of the genome. Another challenge is the delivery of CRISPR to the patient’s cells, which requires efficient methods to get the CRISPR components into the cells and ensure that they reach the correct tissues.

The second approach involves using CRISPR to create healthy blood stem cells that can produce functional beta-globin. This is done by using CRISPR to edit the DNA of stem cells in the lab, so that they produce healthy β-globin protein. The edited cells are then expanded in the lab and transplanted back into the patient, where they can repopulate the patient’s bone marrow and produce healthy blood cells. Hemoglobin F is encoded by a gene distinct from HBB, and is unaffected by the disease-causing mutation. This method edits the disease-causing base substitution in one step. CRISPR is used to correct the hemoglobin E mutation by homology-directed repair using a single-stranded DNA template that includes the normal HBB genetic sequence. The method was implemented in extracted iPSCs, and the presence of the correct sequence was validated by Sanger sequencing.

This approach has shown some success in early clinical trials, but there are still challenges to overcome, such as ensuring that the edited cells engraft successfully in the patient’s bone marrow and produce enough functional blood cells. Without this step ging seamlessly, this procedure could cause serious infections, rejections and even fatality.

Overall, while CRISPR-based therapies for β-thalassemia are still in the experimental stage, they hold promise as a potential curative treatment for this genetic blood disorder. However, more research is needed to address the challenges and ensure the safety and efficacy of these therapies before they can be widely used in patients.

5.0 Clinical trials

Countless clinial trials are taking place on a global scale in order to harness this promising technique. These trials follow the main steps such as hemoglobin insertion but they vary in progression times and infusion levels. Most trials as of now are 1/2 clinical trials showing area for imporvement and growth.

A 1/2 clinical trial is a study that tests the safety, side effects, and determines the best dose of a new treatment. Such trials also test how well a certain type of cancer or other disease responds to a new treatment. In the phase 2 part of the clinical trial, patients usually receive the highest dose of treatment that did not cause harmful side effects in the phase 1 part of the clinical trial. Combining phases 1 and 2 may allow research questions to be answered more quickly or with fewer patients and allows for profficient development and research.

Full success is yet to be seen but the up and coming trials have progressed to an increased amount of cured conditions alongside dimunished mortality.

Below are some predominant trials which uphold are hope of an efficient, accesible sure for β-thalassemia.

5.1 CTX001

CTX001 is a Phase 1/2 clinical trial being conducted by CRISPR Therapeutics and Vertex Pharmaceuticals. It is currently evaluating the safety and efficacy of CRISPR-edited blood stem cells in patients with β-thalassemia as well as sickle cell disease.

In this trial, patients receive an infusion of their own blood stem cells that have been edited using CRISPR to produce functional β-globin. Preliminary results from this trial have been promising, with some patients showing sustained production of functional hemoglobin and reduced need for transfusions.

5.2 EDIT-BTH

EDIT-BTH is another Phase 1/2 clinical trial being conducted by Editas Medicine, which is also evaluating the safety and efficacy of CRISPR-edited blood stem cells in patients with β-thalassemia.

In the EDIT-BTH trial, patients receive an infusion of blood stem cells that have been edited using CRISPR to correct the genetic mutations that cause β-thalassemia. The editing is done ex vivo, meaning the patient’s blood stem cells are extracted from their body and edited in the laboratory before being reintroduced back into the patient.

5.3 LUMINATE

LUMINATE is a Phase 1 clinical trial being conducted by Intellia Therapeutics. In the LUMINATE trial, patients receive an infusion of their own blood stem cells that have been edited using CRISPR to produce functional β-globin. The editing is done ex vivo, meaning the patient’s blood stem cells are extracted from their body and edited in the laboratory before being reintroduced back into the patient.

The editing is performed using Intellia’s proprietary lipid nanoparticle delivery system, which delivers the CRISPR components into the blood stem cells with high efficiency and specificity. The edited blood stem cells are then expanded in the laboratory and reinfused back into the patient’s bloodstream.

The main goal of the LUMINATE trial is to evaluate the safety and tolerability of the CRISPR-edited blood stem cells. Additionally, tassessing the engraftment and expansion of the edited cells, as well as measuring the production of functional β-globin and the reduction in the need for blood transfusions is also being monitored.

6.0 Conclusion

In conclusion, using the CRISPR system in order to combat β-thalassemia is becoming increasingly conspicuous and displays feasible cures for this delibitating disease.

With the continuation of deligent research alongside clinical trials, a future with the cure of β-thalassemia is truly not far of reach.