During the process of correcting mutations via gene therapy, is the defective gene removed?

Just recently started learning about gene therapy, many websites explain that the corrected DNA can be added to the genome using a vector and all that. I just don't understand what happens to the other sequence of nitrogenous bases that codes for the incorrect protein. Thanks in advance for any answers :)

To be specific: I am talking about adult, somatic gene therapy here, and germline gene therapy experiments is still a landmine when considering ethical reasons.

The defective gene codes for a defective protein, that usually plays a part in pathways. Since the protein is also defective, that pathway is also rendered defective because of this protein, and usually when pathways go defective, bad things happen.

There are three target approaches to gene therapy:

  1. Gene silencing by Antisense Oligonucleotide Therapy
  2. Genome Surgery by ZFN, Crispr/Cas9 etc…
  3. Gene Replacement by viral vectors

The only method that corrects the defective gene here is the genome surgery method. By using Zinc Finger Nucleases, or Crispr/Cas9s, TALENS or other methods like that, the defective protein is corrected at the genome level, which automatically corrects the mRNA and protein, and everything works as it should.

Other methods like Antisense, just prevent the incorrect protein from forming. This is really useful if the defective protein is itself toxic to the body, and a replacement protein can be achieved by masking the defective region to produce a truncated but functional protein.

Gene replacement usually puts in a healthy copy of the gene in, that functions along with the defective copy, and this solves majority of the problems as the affected pathway is no longer rendered inactive, as the healthy protein takes over the defective pathway. The defective protein is still produced, but doesnt do anything (or rather, is rendered moot as the healthy protein takes over) and gets removed during regular maintenance of the cell.

Now depending on the type of disease the individual has, gene replacement may also take place along with gene knockout (depending on if the defective gene is toxic), but obviously many considerations have to be thought about before the combinatorial therapy can be used (toxicity of treatment itself being the primary concern, followed closely by efficacy).

Now obvious question with gene replacement, antisense or any other form of gene therapy is how we get the therapeutic agent to all cells, and yes that is a major challenge, and that's why gene therapy is expensive. And no, the therapy doesn't correct all the cells in your affected tissue, but it aims to correct majority of the cells, and that should restore function (atleast in myopathies like the muscular dystrophies)

As you can see here, in DMD, dystrophin gets muated due to a mutation We can either mask the mutation by Antisense therapy at the level of the RNA (which leads to a functional protein forming), put in an alternative form of dystrophin (mini or micro dystrophin) that takes over the function of dystrophin in majority (if we are so lucky) of the cells, and the overall phenotype of the individual improves. Recent advances in genome surgery are also being investigated for ameliorating disease symptoms. The reason I concentrated on DMD is that, gene therapy for DMD is my PhD. So apologies for the bias.

Genetic Disorders & Gene Therapy

Genes are found in chromosomes, and are basic physical and functional units of heredity. They’re specific sequences of bases which code to make proteins. Genes are given importance because of proteins it’s proteins that perform most of the functions of life. Proteins also work to build our numerous cellular structures.(Citation 4)

When genes are altered so that encoded proteins are unable to carry out their normal functions, genetic disorders can result. These alterations can form on several different levels or scales. A point mutation in a gene (i.e. when a single nucleotide is mistakenly altered during synthesis) is not usually serious. For example, Sickle-cell disease, which is an exception because it is in fact a serious disease, is caused by a point mutation on β-globin chain of hemoglobin. Hydrophilic amino acid glutamine acid is replaced with hydrophobic amino acid valine in the sixth position. Mutation occurs on Chromosome 11. In Sickle-cell anemia, the red blood cells turn sickle-shaped, thereby causing problems in blood flow and oxygen-carrying capacity. People who inherit a sickle hemoglobin gene from one parent and a normal gene from the other parent have a condition called sickle cell trait

Other mutations can occur on a larger basis, sometimes, for example, due to the addition of an extra nucleotide in a gene or the removal of the nucleotide. Both of these can shift the entire synthesis reading frame from the point they are removed or added.

Classification of Genetic Disorders

Genetic disorders can be classified on basis of :

Level 1: Single Gene Disorders

Disorders which result when a mutation causes the protein product of a single gene to be altered, differentiated, or missing.

Level 2: Chromosome Abnormalities

In the Level 2 disorders, entire or whole chromosomes, or large segments of the chromosomes, are missing, duplicated, or altered.

Level 3: Multifactorial Disorders

Multifactorial disorders are those which result from mutations in multiple genes. They’re complex, often coupled with environmental causes.(Citation 5)

Genetic disorders are often life-crippling. Most of them are present at birth or sometime early in life. Many of them result in life-threatening situations, mental, physical, or sexual disability, or even eventually fatal turns.

What makes genetic disorders even worse is that some of them can be inherited this means that certain members of the populations can be only carriers for a genetic disease, thus holding the genetic disease without actually experiencing it. Thus, a disease can unknowingly pass on until it affects a generation.

Genetic disease is widely prevalent Cystic fibrosis, for example, is a genetic disease that affects the lungs and digestive system of about 30,000 children and adults in the United States (70,000 worldwide). Approximately 83,000 children and adolescents with Down Syndrome were living in the United States in 2002. Cancer has been shown to have a genetic cause as well, as have many other disorders.

So, the key question is: what can be done about genetic disorders?

This is where gene therapy enters

Gene therapy is defined as a “technique for correcting defective genes responsible for disease development”.(Citation 6)

Gene therapy involves, in its most basic sense, the application of treatment on a genetic level in order to provide therapy or cure for genetic diseases.

There are several different types of approaches to gene therapy. One common approach involves simply replacing a faulty gene with a good gene with the use of manipulated vectors, such as viruses, or even microsurgery, using nanotechnology. Another method involves editing the chromosome itself in order to remove any defective genes. Genes causing a disease can be “switched” on or off. Genes could also be reverse-mutated back into normal genes.

What differentiates gene therapy from other types of therapies? Firstly, gene therapy is a rather more permanent solution. Turning off the gene causing a disease, such as cancer, for example, would be more effective than providing constant non-genetic therapeutics for the cancer. As the creators of the University of Utah’s “Learning Genetics” website write, gene therapy is akin to fixing a broken window: one has the option of either repairing the cracked window with tape, or putting in an entirely new window. In a similar way, gene therapies can involve replacing the faulty gene entirely, thus preventing future occurrences or remissions of the disease. Whereas most drug-based approaches only serve to cure symptoms, gene therapy provides a way to fix a problem at its source.(Citation 7)

A visualization of germ line gene therapy. In germ line gene therapy, note that the healthy child will also pass on the corrective genes to his or her children. At the same time of added benefit, germ line gene therapy is also highly controversial and therefore not as rigorously

Ex-vivo somatic gene therapy. Somatic gene therapy is mostly performed in developed humans and provides the healthy gene for only the patient. His offspring will not carry the corrective gene.

A model of ex-vivo gene therapy. Ex-vivo gene therapy is more commonly used.

Shown on the left side is an in-vivo approach to gene therapy. Here, the therapeutic nucleic acid is inserted directly into the patient. The gene is packaged into one of several types of vectors and delivered with a device to a target organ. In the visual shown, the gene is incorporated into a plasmid and delivered to liver via a catheter in portal vein.

As shown on right side, an ex-vivo approach involves harvesting cells from tissue of interest, transducing them with a gene in vitro, and again administering the genetically altered cells to the patient.

A globular conformation consisting of a 500-kilobase gene-rich domain. It's located on human chromosome number 16. Proteins are vital to functioning proteins are made when genes are synthesized.

Several types of mutations can lead to problems in a gene. Notice how some mutations change the amino acid composition of the synthesized protein.

Neurological diseases caused by triplet repeat amplifications in chromosome locus areas.

A cause of genetic disease: non-disjunction in chromosomes. Normal meiosis is shown in A). B) shows non-disjunction in meiosis I, cellular division process, and (C) shows non-disjunction in meiosis II, the second phase of meiosis.

A potential gene therapy designed and developed by ArmaGen to cross the blood-brain barrier in order to rescue dying nerves.

There are two basic types of gene therapy.

Germ line Gene Therapy - Germ line gene therapy involves altering the genetic makeup of a gene of either an egg or a sperm cell before fertilization, or altering the genetic composition of a blastomere during an early stage of its division.

  • Advantages - Germ line gene therapy is done before the organism has grown or developed therefore, the cure is inherited by future generations of that organism. Also, germ line gene therapy allows for a desired gene to become fully incorporated into the organism before activation therefore, one of the factors influencing unwanted immune responses (to the gene) is removed.
  • Disadvantages - Germ line gene therapy is very controversial. In addition, due to this controversial nature and other limiting factors, germ line gene therapy is not fully pursued for development. Germ line gene therapy also holds numerous risks, such as a margin for possible error during the gene 'transplant'

Somatic Gene Therapy - Somatic gene therapy, unlike germ line gene therapy, involves altering the genetic code or chromosomes of a person's somatic cells, or body cells. It is mostly performed in fully grown organisms.

Mechanisms and Risks of Gene Therapy

Human diseases that result from genetic mutations are often difficult to treat with drugs or other traditional forms of therapy because the signs and symptoms of disease result from abnormalities in a patient’s genome. For example, a patient may have a genetic mutation that prevents the expression of a specific protein required for the normal function of a particular cell type. This is the case in patients with Severe Combined Immunodeficiency (SCID), a genetic disease that impairs the function of certain white blood cells essential to the immune system.

Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce. Viral vectors such as adenovirus are sometimes used to introduce the functional gene part of the viral genome is removed and replaced with the desired gene (Figure 1). More advanced forms of gene therapy attempt to correct the mutation at the original site in the genome, such as is the case with treatment of SCID.

Figure 1. Gene therapy using an adenovirus vector can be used to treat or cure certain genetic diseases in which a patient has a defective gene. (credit: modification of work by National Institutes of Health)

So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosis and adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

Gene Therapy Gone Wrong

The risks of gene therapy were realized in the 1999 case of Jesse Gelsinger, an 18-year-old patient who received gene therapy as part of a clinical trial at the University of Pennsylvania. Jesse received gene therapy for a condition called ornithine transcarbamylase (OTC) deficiency, which leads to ammonia accumulation in the blood due to deficient ammonia processing. Four days after the treatment, Jesse died after a massive immune response to the adenovirus vector. [1]

Until that point, researchers had not really considered an immune response to the vector to be a legitimate risk, but on investigation, it appears that the researchers had some evidence suggesting that this was a possible outcome. Prior to Jesse’s treatment, several other human patients had suffered side effects of the treatment, and three monkeys used in a trial had died as a result of inflammation and clotting disorders. Despite this information, it appears that neither Jesse nor his family were made aware of these outcomes when they consented to the therapy. Jesse’s death was the first patient death due to a gene therapy treatment and resulted in the immediate halting of the clinical trial in which he was involved, the subsequent halting of all other gene therapy trials at the University of Pennsylvania, and the investigation of all other gene therapy trials in the United States. As a result, the regulation and oversight of gene therapy overall was reexamined, resulting in new regulatory protocols that are still in place today.

Think about It


With the Food and Drug Administration’s trio of gene therapy approvals in 2017, the innovation floodgates have opened. Pharmaceutical and biotechnology companies are seeking to capitalize on a revolutionary therapeutic approach that was once deemed too risky. With promising clinical results from early gene therapies, venture capital money is finally freeing up to enable this exciting next generation of medicine.

However, even the most well-funded gene therapy programs are exceedingly complex to navigate through preclinical development. These safety and efficacy tests, which are required before drugs can be studied in humans, are perhaps the most perilous stage of the entire development process. Drug companies must design studies that demonstrate, with sound data, that the therapy is safe and effective before regulators deem it suitable for dosing in patients.

The stakes couldn’t be higher. Depending on the outcome of preclinical safety and biodistribution studies, investigational gene therapies will either move to the final stage of FDA review or they’ll fizzle, sending scientists back to square one.

Lovelace Biomedical has been conducting safety and biodistribution studies on investigational gene therapies for more than a decade, making it one of the country’s longest-running gene therapy programs in the preclinical space. With recent gene therapy projects in cystic fibrosis, Pompe disease and a variety of other rare disorders with a critical patient need, the Lovelace team understands the nuances of designing and implementing a preclinical program supporting an IND submission that answers all regulatory questions.
Here, with input from across the Lovelace gene therapy team, we provide an inside look at the steps most drug sponsors must take as they advance a potential gene therapy through preclinical studies for eventual regulatory approval. One thing is clear: There is no cookie cutter path for gene therapy products. In an emerging therapeutic space, every step requires a customized and highly informed approach.

“I believe gene therapy will become a mainstay in treating, and maybe curing many of our most devastating and intractable illnesses.” Scott Gottlieb, M.D. – FDA Commissioner, Dec. 2017

How will the corrective DNA at the core of a gene therapy be delivered into patients’ cells? Typically, the answer is through a virus. Adeno associated viruses (AAV), adenoviruses and lentiviruses are most commonly used as vectors for drug delivery. This choice hinges on therapeutic approach: AAV vectors provide long-term gene expression and naturally occurring sub types (serotypes), which allows for some target-tissue specificity. Adenovirus vectors may be optimal when short-term gene expression is the goal. And lentiviruses are most often used to transfer genetic material to patient cells in culture (bone marrow or blood cells) that are subsequently injected back into the patient as therapy to treat immune deficiencies or sickle cell anemia.

Other modes of gene delivery include liposomes (lipid particles) and nanoparticles that can be engineered to target specific cell types for delivery. The advantage with these is that they do not produce immune responses that are associated with viral vector use.

“You have to get the vector to the tissue, convince the cells to incorporate the vector, and then get the genetic material to express the gene,” says Lovelace Chief Scientific Officer Jake McDonald. “For most of the gene therapies in development, which seek to provide a curative treatment for hereditary conditions, you want patients to express this gene for the rest of their lives.”

The route of drug administration depends on the target organ or tissue where the defective gene is expressed. For example, with ocular, joint or brain-directed therapies, the vector is delivered directly to the eye, joint, or brain or spinal fluid. In the case of Pompe disease, which damages muscle and nerve cells throughout the body, the vector s given either intravenously or into the diaphragm or skeletal muscle. For cardiac therapies, the drug may be administered via catheter to the coronary vessels, or directly applied to the surface of the heart. For Alpha-1 antitrypsin deficiency, a genetic disorder that leads to lung and liver problems, the vector is administered intravenously or into the pleural space between ribs and the lung.

As for the timing of the therapy, this too is an important consideration for preclinical studies. With inherited diseases, it’s optimal to deliver the medicine to the fetus or infant as soon as possible after diagnosis. However, with few exceptions, the FDA requires testing in adults or children at least 12 years old before allowing delivery to babies or small children.

“These decisions are critical, as patients only have one chance at gene therapy,” McDonald said. “After the body is exposed to the new genetic material a second time, the immune system will reject it. New technologies are under development that may avoid this challenge, but that has been the situation to date.”

Except for instances in which vector is administered to confided spaces such as the eye or joint, experience has shown that gene therapy will distribute to off- target sites. However, this unwanted effect can be diminished by incorporating “promoters” within the vector to limit or control gene expression. Some unique promoters have sensitivity to light or oxygen tension to control gene expression. With Pompe disease, a desmin-specific promotor limits expression to a limited tissue set, including muscle, although the AAV vector distributes through the blood to most of the body’s tissues.

Choosing a species for preclinical testing of gene therapies is one of the most challenging decisions of study design. That’s because most gene therapies seek to treat rare diseases, which can be difficult or impossible to replicate in an animal.

Some models will have a naturally occurring mutation, while others are genetically modified (as seen in the GAA knockout model of Pompe disease or Sandoff mouse model of Tay Sachs disease). Animal models may be developed chemically, as with mono-iodoacetate-induced osteoarthritis, or through physical means, as with cardiac failure in pigs induced by vascular occlusion or electrical pacing. In addition, the sponsor may conduct in vitro studies to demonstrate to the FDA that a given therapy will be taken up by cells of a chosen species in a manner similar to uptake in human cells, or that the receptors being targeted in human cells are also present in the animal species.

“There can be many layers of complication influencing the drug sponsor’s decision here,” McDonald said. “You’re delivering a human gene to an animal. And an animal may or may not respond to the human protein in the same way it would to a protein from its own species. That is one reason why a company may decide to evaluate its therapy in two species, such as mouse and nonhuman primate.”
The rationale for the species used must be justified in the pre-IND package and in the IND — and this is something for which Lovelace brings vast insight. For example, while nonhuman primates can serve as disease model for certain conditions, many primates naturally have some level of neutralizing antibodies to the vector that’s used to deliver the gene therapy — which means the animal would show no response to a gene therapy. For this reason, all nonhuman primates must be prescreened.

At this stage of research, it’s essential to have top veterinary talent on your side. When measuring disease and response to treatment in an animal model, the team must be able to distinguish between the disease itself and the toxicological effects of the treatment, which requires skill and experience. In some cases, scientists evaluate efficacy and safety at the same time and in the same model.

Many factors are taken into account when designing the preclinical study, from number of dose groups, number of animals per dose group, types of controls, and number of endpoint-sampling time points. Unless a genetic disease occurs in only one sex, both sexes are included in safety and biodistribution studies. In most cases, at least two vector doses are used. Multiple sampling time points are included, beginning at the point when vector expression is to peak (usually 7 to 14 days) and extending for several months to one year.

Common endpoints for a gene therapy study include: body weight, clinical signs, hematology, serum chemistry, vector biodistribution (as evaluated by PCR), gene expression in target tissue and in tissues having a pre-specified large concentration of vector capsid (the shell of the virus), neutralizing antibodies in serum to capsid protein and transgene, immune responses (T cell-mediated to capsid protein and expressed protein), and histopathology (and immunohistochemistry for microscopic evaluation of gene expression). Other endpoints may be included, depending on the disease. Another key point to note here: If the drug sponsor will be seeking regulatory approval in Europe, an additional step may be required to evaluate vector concentration in bodily fluids and excreta to determine shedding.

After preclinical results are evaluated using the latest bioanalytical tools and reporting, it’s time to complete the IND application — showing the strong data that indicates your drug is ready for testing in patients. All preclinical safety studies for gene therapies are conducted under Good Laboratory Practice (GLP) guidelines. An audited final report with summary data, statistics and appendices containing contributing scientist reports and individual animal data is submitted to the sponsor for inclusion in the IND package.


Beta-thalassemia (β-thal) and sickle cell disease (SCD), two of the most common genetic diseases, are caused by mutations in the HBB gene encoding the postnatal form of the beta subunit of hemoglobin. After birth, hemoglobin tetramers contain two alpha subunits and two beta globins coded by the HBB gene that is expressed neonatally and after. Before that, beta globins coded by one of the two HBG genes that are expressed during the fetal stage and normally silenced after birth. While a point mutation in codon 6 (GAG > GTG, resulting in substitution of glutamic acid to valine amino acid) in the HBB gene creates a SCD trait, various mutations in HBB gene resulting in reduced or absent of HBB protein cause β-thal starting in early childhood. Over 200 different types of mutations in the HBB gene have been identified in patients with β-thal, which could be located anywhere within the ∼1,600 basepair (bp) DNA segment containing the three coding exons, splicing sites, and other regulatory elements 1 . Patients with mutations in both HBB alleles that significantly reduce the HBB protein production (called β-thal major or Cooley's anemia) suffer from severe anemia and skeletal abnormalities, and have a high level of mortality or shortened life expectancy if left untreated 1 . Similarly, patients carrying both copies of the SCD HBB mutation, or a heterozygous SCD mutation plus a copy of a severe β-thal mutation will make dysfunctional HBB protein that impedes hemoglobin functions 1 .

Although chronic transfusion of red blood cells and some small molecules ameliorate symptoms of β-thal and SCD patients, it is highly desirable to develop a cure for treating these monogenic diseases due to HBB gene mutations. Bone marrow transplantation (BMT) using hematopoietic stem cells (HSCs) from an allogeneic donor with the wildtype HBB gene has been explored in the past several decades for treating β-thal and SCD. Although successful in some cases, the BMT technology is limited because of graft-versus-host disease and a lack of immunologically matched donors that are unrelated to the treated patients 2 . An alternative approach is to insert a functional copy of the HBB gene into the patient's HSCs followed by BMT. In the past decades, scientists have overcome many hurdles in efficient delivery of a functional copy of the HBB gene ex vivo into human HSCs, which will home into patient's marrow, differentiate to erythrocytes and express a high-level of the added HBB gene 2, 3 . Currently, the best developed approach of gene therapy for treating β-thal and SCD patients relies on using genome-inserting lentiviral vectors that carry the HBB or related HBG coding sequence (CDS) plus shortened regulatory elements, inserting them permanently into the genome of autologous HSCs 2-4 . Although ongoing clinical trials will ultimately determine the balance of efficacy and risks for treating β-thal and SCD patients, the uncontrollable nature of lentiviral vector insertion that favors coding regions is always a potential risk especially over a long-term 2-7 . In recent years, scientists moved back to achieve precise genome editing via homology-directed repair (HDR) of a HBB mutation, which has been explored since 1985 but with a very low efficiency (10 −6 ) 7, 8 .

The recent advents of engineered nucleases that make a double-stranded DNA break (DSB) greatly improved our ability to achieve HDR and other forms of DNA repair and recombination in nontransformed human cells. In addition, the availability of immortalized human stem cells harboring HBB mutations with ability to differentiate to erythrocytes significantly accelerates the development of functional correction of HBB mutations. Since 2008, it became possible to generate human induced pluripotent stem cells (iPSCs) from β-thal and SCD patients that have unique HBB mutations 9-12 . During this time, engineered nucleases such as Zinc Finger Nucleases, Transcription Activator Like Effector Nucleases and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems have also been developed to enhance HDR and achieve precise genome editing to correct a HBB mutation in iPSCs 6-8 . Although the HDR efficiency is still relatively low (<1%) in nontransformed cells, rare clones of iPSCs after HDR-mediated gene correction can be selected, characterized, and expanded extensively. Aided by validated nucleases targeting specific locations of various specific HBB mutations, precise genome correction of the SCD point mutation in exon 1, the TCTT deletion in exon 2, or the IVS2-654 mutation in intron 2 has been achieved when a donor DNA template specific for each type HBB mutation is also provided 13-23 . The ease and robustness of the CRISPR/Cas9 system has become the preferred choice in recent years for making a specific DSB in the HBB locus and achieving HDR to correct a specific HBB gene mutation 17-23 .

For future clinical applications of correcting various HBB mutations, it is highly desirable to develop a universal strategy to correct most if not all of >200 types of HBB mutations by using validated CRISPR guide RNAs and one donor DNA template for HDR. To provide a proof-of-principle, we developed a strategy of using two validated guide RNAs (targeting at the HBB exon 1 and 3′-un-translated region (UTR)) and a DNA template providing all the HBB CDS. In this way, a HDR event near the guide RNA will provide a functional correction of HBB mutations not only in exon 1, but also exon 2 and 3 or any downstream sites. We used iPSC lines from two transfusion-dependent β-thal patients with HBB mutations in exon 2 and intron 2 as well as an exon 1 mutation to test this new and more universal strategy. To provide a simple readout, we linked a GFP reporter gene downstream to the HBB coding cDNA via the 2A self-cleaving peptide so that the GFP reporter expression is indicative of the HBB expression from the same transcript and pro-peptide. Our data provide evidence that this universal approach is able to correct various HBB gene mutations and restore HBB protein production. In addition, it provides an experimental system to screen bioactive molecules and to improve HBB protein expression in iPSC-derived erythrocytes based on coexpression of GFP reporter.

Gene therapy of hematological disorders: current challenges

Recent advances in genetic engineering technology and stem cell biology have spurred great interest in developing gene therapies for hereditary, as well as acquired hematological disorders. Currently, hematopoietic stem cell transplantation is used to cure disorders such as hemoglobinopathies and primary immunodeficiencies however, this method is limited by the availability of immune-matched donors. Using autologous cells coupled with genome editing bypasses this limitation and therefore became the focus of many research groups aiming to develop efficient and safe genomic modification. Hence, gene therapy research has witnessed a noticeable growth in recent years with numerous successful achievements however, several challenges have to be overcome before gene therapy becomes widely available for patients. In this review, I discuss tools used in gene therapy for hematological disorders, choices of target cells, and delivery vehicles with emphasis on current hurdles and attempts to solve them, and present examples of successful clinical trials to give a glimpse of current progress.

Gene therapy techniques

There are several techniques for carrying out gene therapy. These include:

Gene augmentation therapy

  • This is used to treat diseases caused by a mutation that stops a gene from producing a functioning product, such as a protein.
  • This therapy adds DNA containing a functional version of the lost gene back into the cell.
  • The new gene produces a functioning product at sufficient levels to replace the protein that was originally missing.
  • This is only successful if the effects of the disease are reversible or have not resulted in lasting damage to the body.
  • For example, this can be used to treat loss of function disorders such as cystic fibrosis by introducing a functional copy of the gene to correct the disease (see illustration below).

Gene inhibition therapy

  • Suitable for the treatment of infectious diseases, cancer and inherited disease caused by inappropriate gene activity.
  • The aim is to introduce a gene whose product either:
    • inhibits the expression of another gene
    • interferes with the activity of the product of another gene.

    Killing of specific cells

    • Suitable for diseases such as cancer that can be treated by destroying certain groups of cells.
    • The aim is to insert DNA into a diseased cell that causes that cell to die.
    • This can be achieved in one of two ways:
      • the inserted DNA contains a “suicide” gene that produces a highly toxic product which kills the diseased cell
      • the inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system.

      Gene Therapy for Beta-Hemoglobinopathies: Milestones, New Therapies and Challenges

      Inherited monogenic disorders such as beta-hemoglobinopathies (BH) are fitting candidates for treatment via gene therapy by gene transfer or gene editing. The reported safety and efficacy of lentiviral vectors in preclinical studies have led to the development of several clinical trials for the addition of a functional beta-globin gene. Across trials, dozens of transfusion-dependent patients with sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) have been treated via gene therapy and have achieved reduced transfusion requirements. While overall results are encouraging, the outcomes appear to be strongly influenced by the level of lentiviral integration in transduced cells after engraftment, as well as the underlying genotype resulting in thalassemia. In addition, the method of procurement of hematopoietic stem cells can affect their quality and thus the outcome of gene therapy both in SCD and TDT. This suggests that new studies aimed at maximizing the number of corrected cells with long-term self-renewal potential are crucial to ensure successful treatment for every patient. Recent advancements in gene transfer and bone marrow transplantation have improved the success of this approach, and the results obtained by using these strategies demonstrated significant improvement of gene transfer outcome in patients. The advent of new gene-editing technologies has suggested additional therapeutic options. These are primarily focused on correcting the defective beta-globin gene or editing the expression of genes or genomic segments that regulate fetal hemoglobin synthesis. In this review, we aim to establish the potential benefits of gene therapy for BH, to summarize the status of the ongoing trials, and to discuss the possible improvement or direction for future treatments.

      Conclusions and perspectives

      The last few decades witnessed a revolution in the development and application of gene therapy. There is currently no doubt that gene modification approaches have turned into a valuable biotechnology and therapeutic tool. New and safer vector designs along with a better comprehension of vectors biology led to successful utilization of these valuable tools in several clinical contexts now. The success of retro and lentivirus-based gene therapies helped to turn gene therapy into a solid and flourishing field. Non-viral integrative vectors, such as transposons, have the potential to extend this success story, hopefully making gene therapy approaches more straightforward, simple and cost-effective.

      The newly developed genome-editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered regularly-interspaced short palindromic repeats (CRISPRs) represent the most recent tools for genetic manipulation. Even if clinical safety of these tools are still to be clarified and there is undoubtedly still room for the improvement of such approaches, the ability to edit specific genome sequences could revolutionize the whole cell biology, biotechnology, cell engineering and gene therapy areas. Such tools may allow approaches such as add back of gene function, site-directed gene corrections and gene replacements, impacting activities such as animal transgenesis and the incipient logic-systems and biological fields. Hopefully the combination of gene delivery approaches such as those described in this review with the new gene editing tools will turn gene therapy into a more effective and curative approach.