Possible complications due to gene-therapy?

Possible complications due to gene-therapy?

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Edward Lanphier, CEO of Sangamo BioSciences mentions here( that scientists can now specifically target cells, change the genome within a cell. Say you want to cure a type of disease, which can be linked to your genome. You have successfully removed the faulty gene in your genome (GenomeA), replaced it with a correct gene resulting in a improved genome (GenomeB) in a single cell. The next step will probably be to put it back into the human body. However, as I understand it every human cell contains your faulty human genome (GenomeA).


  1. Even if you put a few cells with the correct genome sequence (GenomeB) into your human body, it is probably going to be overwhelmed by the other cells that contain your faulty genome (GenomeA). Cells containing the correct genome (GenomeB) will divide and multiply but so will cells containing the faulty genome (GenomeA). In this “genomic war” will the fewer cells containing the correct Genome (genomeB) ever win? Or will you need period infusions of cells with the correct genome "forever"?

How would you guys think about this?

Optional questions:

  1. Even if it wins, it will probably take a very long time to completely replace all the cells with the faulty genomes. How much time is required before you can completely replace all the cells with the incorrect genomeA, with cells containing the correct genomeB?

  2. Say you realize that you have a faulty genome (GenomeA), and you attempt to replace your genome with a correct genome (GenomeB). After 2 years, you have another disease and realize that you have to again correct your genome with GenomeC. However, 2 years is not enough to replace all your cells with the incorrect GenomeA, with the correct GenomeB. If you attempt to undergo genetherapy, now you will have to introduce another group of cells with GenomeC. Now you have cells with multiple different types of genomes floating around: genomeA, genomeB and genomeC. Are there any complications that happen due to the presence of multiple types of cells with different genomes in your body?

Your question is too broad. It is better to take some specific example. Not all cell's in the body divide. If you take cell out of the body and want to return it back, it must divide. So we probably could talk about stem cells for example. The stem cells don not really sit in one place. And it almost impossible now to get such cells from the place that they sit in it, and then return in back. It is possible with bone marrow for example.

If we take some example, like ALS - some percent of the disease is thought to be genetic. But the mutation is in all the body cells. Mostly it's affects astrocytes, microglia and motor neurons. You cant take them out of the body, fix and return back. The only chance to fix is to work inside the body. For example by using some sort of virus (AAV for ex.) And CRISPR.

However, if we follow your story and we insert cells with genomB, they will live together with cells with genomA, if there is not some bigger mortality of the original cells.

New gene delivery method: Magnetic nanoparticles

Stent angioplasty saves lives, but there often are side effects and complications related to the procedure, such as arterial restenosis and thrombosis. In the June 2013 issue of The FASEB Journal, however, scientists report that they have discovered a new nanoparticle gene delivery method that may overcome current limitations of gene therapy vectors and prevent complications associated with the stenting procedure. Specifically, this strategy uses stents as a platform for magnetically targeted gene delivery, where genes are moved to cells at arterial injury locations without causing unwanted side effects to other organs.

Additionally, magnetic nanoparticles developed and characterized in the study also protect genes and help them reach their target in active form, which also is one of the key challenges in any gene therapy.

"This study can help address a number of barriers to translation of experimental gene therapeutic approaches to clinical practice," said Michael Chorny, Ph.D., a researcher involved in the work from the Division of Cardiology at the Abramson Pediatric Research Center at The Children's Hospital of Philadelphia in Pennsylvania. "Bringing gene therapy closer to clinical use is a step toward developing safer and more effective ways for treating cardiovascular disease."

To make this technique possible, Chorny and colleagues used in vitro vascular cells to demonstrate the ability to effectively deliver genes using biocompatible nanoparticles and magnetic force without causing adverse effects. Although effective gene transfer in these cells has been difficult to achieve historically, this study demonstrated that magnetically guided "gene-impregnated" nanoparticles delivered their cargo effectively, especially compared to conventional gene delivery vectors. Next, researchers explored magnetically targeted gene delivery by applying these nanoparticles to stented arteries in rats. The nanoparticle-mediated expression of stent-targeted genes was shown to be greatly enhanced in treated animals when compared to control groups treated with nanoparticles without using the magnetic conditions, or with an equivalent dose of a conventional gene delivery vector. Genes delivered using the magnetically targeted nanoparticles were also expressed at considerably higher levels in the stented arteries compared to other organs and tissues.

Gene Therapy Restores Immune Function in Children with Rare Immunodeficiency

A DNA double helix rests on a print-out illustration of the DNA letters A, T, C and G.

A DNA double helix rests on a print-out illustration of the DNA letters A, T, C and G.

An investigational gene therapy can safely restore the immune systems of infants and children who have a rare, life-threatening inherited immunodeficiency disorder, according to research supported in part by the National Institutes of Health. The researchers found that 48 of 50 children who received the gene therapy retained their replenished immune system function two to three years later and did not require additional treatments for their condition, known as severe combined immunodeficiency due to adenosine deaminase deficiency, or ADA-SCID. The findings were published today in the New England Journal of Medicine.

ADA-SCID, which is estimated to occur in approximately 1 in 200,000 to 1,000,000 newborns worldwide, is caused by mutations in the ADA gene that impair the activity of the adenosine deaminase enzyme needed for healthy immune system function. This impairment leaves children with the condition highly susceptible to severe infections. If untreated, the disease is fatal, usually within the first two years of life.

“These findings suggest that this experimental gene therapy could serve as a potential treatment option for infants and older children with ADA-SCID,” said Anthony S. Fauci, M.D., director of NIH’s National Institute of Allergy and Infectious Diseases (NIAID). “Importantly, gene therapy is a one-time procedure that offers patients the hope of developing a completely functional immune system and the chance to live a full, healthy life.”

People with ADA-SCID can be treated with enzyme replacement therapy, but this treatment does not fully reconstitute immune function and must be taken for life, usually once or twice weekly. Transplants of blood-forming stem cells, ideally from a genetically matched sibling donor, can provide a more lasting solution. However, most people lack such a donor. Additionally, stem cell transplants carry risks such as graft-versus-host disease and side effects from chemotherapy medications given to help the donor stem cells establish themselves in the patient’s bone marrow.

The new research evaluated an experimental lentiviral gene therapy designed to be safer and more effective than previously tested gene-therapy strategies for ADA-SCID. This gene therapy involves inserting a normal copy of the ADA gene into the patient’s own blood-forming stem cells. First, stem cells are collected from the patient’s bone marrow or peripheral blood. Next, a harmless virus is used as a “vector,” or carrier, to deliver the normal ADA gene to these cells in the laboratory. The genetically corrected stem cells then are infused back into the patient, who has received a low dose of the chemotherapy medication busulfan to help the cells establish themselves in the bone marrow and begin producing new immune cells.

The experimental gene therapy, developed by researchers from the University of California, Los Angeles (UCLA) and Great Ormond Street Hospital (GOSH) in London, uses a modified lentivirus to deliver the ADA gene to cells. Previous gene-therapy approaches for ADA-SCID have relied on a different type of virus called a gamma retrovirus. Some people who have received gamma retroviral gene therapies have later developed leukemia, which scientists suspect is due to the vector causing activation of genes that control cell growth. The lentiviral vector is designed to avoid this outcome and to enhance the effectiveness of gene delivery into cells.

The results come from three separate Phase 1/2 clinical trials, two conducted in the United States and one in the United Kingdom. The U.S. trials, led by principal investigator Donald Kohn, M.D., of UCLA, enrolled 30 participants with ADA-SCID ranging in age from 4 months to 4 years at UCLA Mattel Children’s Hospital and the NIH Clinical Center in Bethesda, Maryland. The U.K. study, conducted at GOSH and led by principal investigator Claire Booth, M.B.B.S., Ph.D., enrolled 20 participants ranging in age from 4 months to 16 years. Most participants acquired and retained robust immune function following gene therapy—96.7% after two years in the U.S. studies and 95% after three years in the U.K. study—and were able to stop enzyme replacement therapy and other medications. Of the two participants for whom gene therapy did not restore lasting immune function, one restarted enzyme replacement therapy and later received a successful stem cell transplant from a donor, and the other restarted enzyme replacement therapy.

The lentiviral gene therapy appeared safe overall, although all participants experienced some side effects. Most of these were mild or moderate and attributable to the chemotherapy that the participants received.

Researchers observed similar outcomes in all three trials, although there were some differences between the studies. Stem cells were collected from bone marrow in the U.S. trials and from peripheral blood in the U.K. trial. In one of the U.S. trials, 10 children were treated with genetically corrected stem cells that had been frozen and later thawed. The two other trials used fresh stem cell preparations. In the future, the freezing procedure—known as cryopreservation—may allow stem cells to be more easily transported and processed at a manufacturing facility far from the patient’s home and shipped back to a local hospital, reducing the need for patients to travel long distances to specialized medical centers to receive gene therapy. A trial of the cryopreserved treatment is now underway at the Zayed Centre for Research into Rare Diseases in Children in London, in partnership with GOSH.

For more information about the trials described in the New England Journal of Medicine paper, visit under identifiers NCT01852071, NCT02999984 and NCT01380990. The investigational lentiviral gene therapy, which is licensed to Orchard Therapeutics, has not been approved for use by any regulatory authority.

The research was funded in part by three NIH Institutes: NIAID the National Heart, Lung and Blood Institute and the National Human Genome Research Institute. Additional funding was provided by the California Institute for Regenerative Medicine, the Medical Research Council, the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children National Health Service Foundation Trust and University College London, and Orchard Therapeutics.

DB Kohn, C Booth et al. Autologous ex vivo lentiviral gene therapy for adenosine deaminase deficiency. New England Journal of Medicine DOI: 10.1056/NEJMoa2027675 (2021).

Principles of angiogenesis

Angiogenic processes are defined as the formation of new vessels. To date, at least three processes contribute to the growth of new blood vessels: angiogenesis, vasculogenesis and arteriogenesis. 30, 31, 32 Angiogenesis – formation of new capillary blood vessels from existent microvessels – occurs in physiological and pathological states. It is a multistep process depending on a multitude of angiogenic and antiangiogenic molecules that plays a crucial role in the development of vascular supply in normal tissue, for example, in reproduction and wound healing, as well as in pathology. Inefficient angiogenesis is involved in many ischemic diseases like heart disease, peripheral vascular disease, rheumatoid arthritis, and tumor growth and metastasis. Apart from angiogenesis, vasculogenesis is known as the development of new vessels from blood stem cells and this process is well recognized in embryogenesis. Blood vessels are derived from mesoderm by differentiation of angioblasts. The third known angiogenic process is arteriogenesis, which refers to the formation and modelling of new arteries possessing fully organized tunica media. Often, this process is described as the maturation of pre-existing collaterals.

Angiogenic processes formulate a complex cascade of events, 30, 31, 32 controlled by a multitude of positive and negative regulators (Table 1). Apart from angiogenic cytokines, many of the extracellular enzymes such as metalloproteinases facilitate angiogenesis by extracellular matrix remodelling. 33, 34 Traditionally, angiogenesis starts with injured tissues (or tumor) that produce and release angiogenic factors that bind to specific receptors located on the endothelial cells of pre-existing blood cells (for VEGF there are VEGFR1, VEGFR2 and VEGFR3 16, 35 ) and activate them to proliferation and migration. The activated endothelial cells migrate to the source of proangiogenic factors (injured tissues, tumor). In the final step, sprouting endothelial cells roll up to organize a blood vessel tube, loops and, finally, matured vessels, stabilized mainly by smooth muscle cells.


Advances and challenges of myogenic cell transplantation

The first trials of transplantation of myogenic cells were done about forty years [7]. Whether it is allogeneic, autogenic or even xenogeneic cells, the transplantation of myogenic cells in animal models or in humans aims to achieve a triple objective.

1) To allow fusion of transplanted myogenic cells with the host muscle fibers. The fusion with the muscle fibers is at the root of the genetic complementation phenomenon in which the myogenic cells of the donor (having the wild gene) provide a normal gene to compensate for the mutated patient gene. There is a genetic mosaicism where healthy nuclei co-exist in the same muscle fibers with the mutated nuclei [16].

2) To allow the formation by the transplanted myogenic cells of new muscle fibers, which must occupy spaces where muscle tissue has been destroyed and replaced with non-functional connective or fatty tissue [17].

3) To generate new satellite cells, the cells which are the main source to regenerate the muscle tissue following damage (Figure 1) [18,19].

Figure 1: Diagram of transplantation of allogeneic myoblasts by intramuscular route. a) Injection of allogeneic myoblasts into the patient's muscle with a needle. b) The injected myoblasts migrate to the perimysium space due to the chemotactic gradient. c) The regeneration of part of the patient muscle fiber (blue). d) Muscle regeneration occurs in the necrotic part of the patient's muscle fiber. e) The regenerated part of the muscle fiber express proteins coded by the exogenous nuclei. f) The transplanted myoblasts also form new satellite cells. g) The grafted myoblasts can fuse together. h) This results in the formation of new muscle fibers.

It should be noted that satellite cells may not be the only myogenic cells capable of regenerating muscle tissue. Other cells such as myoendothelial cells [20], mesoangioblasts [21], pericytes [22], CD33 + [23] and recently cells with aldehyde dehydrogenase activity (ALDH) + [24], interstitial cells PW1+/Pax7- [25] and β-4-integrin+ cells [26] have also been reported to participate in muscle regeneration following their transplantation. Recent advances in cell reprogramming may also be an important source of myogenic cells obtained by the differentiation of iPSC [27].

However, to achieve this threefold objective, several obstacles have to be overcome. These include obtaining and processing the cells to be transplanted, selecting the best route of transplantation, controlling the immune response of the host following the transplantation of allogeneic or xenogeneic cells and developing methods to monitor the transplantation success. The ultimate goal of this approach is the development of a rigorous transplantation protocol in order to ensure successful transplantation on one hand and, on the other hand, to allow the recipient to benefit from it without aggravating his health. In the next section, we will examine the various points listed above.

Obtaining and processing the cells to be transplanted

Most of the myogenic cells used in transplantation are obtained from biopsies of the skeletal muscles of donors. The samples are taken by a surgeon following a protocol approved by the research ethics committee [28]. Biopsies are treated with proteolytic enzymes (collagenase and trypsin) that degrade connective tissue and release satellite cells. The latter are then cultured in vitro to ensure their proliferation as myoblasts. Two major constraints must be overcome at this stage: avoid contamination of successive cell cultures by microorganisms (bacteria, fungi, endotoxin and especially viruses) and maintain the cells at the myoblast stage avoiding their fusion as myotubes.

Selecting the best route of transplantation

There are only two routes of administration of myogenic cells that have been investigated: intramuscular and intraarterial [21,28]. Before examining the two routes of administration, it seems useful to review the pre-treatment of the host muscles before transplantation. Pre-treatment aims to improve the success rate of transplantation by promoting the migration and fusion of myogenic cells with the host muscle fibers. There are two pre-treatments that have been mainly tested: the use of phospholipases [29] and the introduction of intense muscle activity in the animal model [30]. These treatments are aimed at damaging the host muscle fibers. To remove host satellite cells in mouse models, the muscles are either irradiated or cryo-damaged [31,32].

The intramuscular route involves injecting the myogenic cells with a needle mounted on a syringe. It has the advantage of directly bringing the transplanted cells into the host muscular tissue [28,30]. This contribution can, in the best cases, allow the in situ differentiation of the myogenic cells and their fusion with the host muscle fibers located close to transplanted cells. While the approach is effective in animal models with small muscles, such as mice and rats, it is very laborious in nonhuman primates and in humans with larger muscles. Muscle size is not the only obstacle to this approach. The accessibility of certain affected muscles such as the diaphragm, a muscle involved in breathing is a limiting factor in the intramuscular route.

The systemic or intraarterial approach may, in theory, resolve most of the difficulties associated with the intramuscular route. However, it faces a major difficulty. Indeed, according to some studies, the vast majority of myogenic cells (except perhaps mesoangioblasts and CD133+ cells) cannot be extravasated after systemic administration [21,33]. This could be explained by the fact that the myogenic cells do not have a deformable cytoskeleton similar to that of the red blood cells, which can give them the capacity to circulate inside capillaries having a diameter less than their own. This would also allow myogenic cells to cross the junctions of endothelial cells at the level of the capillaries to leave the circulation and reach the muscular tissue where they must differentiate and fuse with the patient muscle fibers. This difficulty may explain the use of the intramuscular route to the detriment of the systemic pathway in the vast majority of myogenic cell transplantation trials.

Controlling the immune response of the host following the transplantation of allogeneic or xenogeneic cells

In almost all transplants, transplanted myogenic cells are allogenic (healthy mice to an mdx mouse, non-human primate to another non-human primate and healthy human to a dystrophic human) and, to a lesser extent, xenogeneic (human, dog or pig myoblasts to a mouse) [19]. In both cases, the immune system of the recipient should be either nonfunctional or permanently suppressed by immunosuppressive drugs to allow for successful transplantation. Some investigators have used cyclosporin A as an immunosuppressive drug [34]. Although immunosuppressive during cell transplants in mice, cyclosporin reduces the fusion of transplanted myoblasts with the host muscle fibers by blocking cell differentiation and inducing apoptosis at therapeutic doses.

Transplantation trials with tacrolimus (FK 506) in mice showed good fusion of myoblasts with the host muscle fibers [35]. This immunosuppressant was also used during myoblast transplantations in non-human primates and in clinical trials with excellent results [28]. To date, tacrolimus remains the only immunosuppressant used in monotherapy in transplantation of myogenic cells because of its low toxicity at therapeutic doses to the recipient and to transplanted myogenic cells. Beyond the use of immunosuppressive drugs, the development of immune tolerance in the host remains a possible alternative [36]. Several studies are being carried out in this direction to solve the problem posed by the long-term use of immunosuppressants. Immuno-deficient mice (nude, rag or SCID) have also been used for transplantation [37-40].

One solution to the problem posed by the rejection of transplanted allogeneic myogenic cells is the use of genetically corrected autogenous myogenic cells. We will address this aspect of the problem in the section on gene therapy.

Developing methods to monitor the transplantation success

The transplantation monitoring has to be done at the short and long term. At short term, the follow-up aims to evaluate the early mortality of transplanted cells. Several studies [19,28] confirmed that approximately 70-80% of transplanted myogenic cells die within 72 hours of transplantation. This high mortality, although it does not prevent transplantation as a therapeutic approach, leads to the necessity to transplant more cells to the DMD patients. The mechanisms underlying this high mortality have not yet been clearly elucidated. Myoblasts that pass from an in vitro (culture) state to an in vivo (recipient’s muscle) state are likely to experience a number of stresses that seriously affect their survival in the new environment. Some studies have identified the expression of pro-apoptic and necrotic factors (intracellular Ca2 + deposition) by transplanted cells that could explain early death by apoptosis [45]. However, these different studies did not explain the upstream factors that activate the different cell death pathways. A careful study of the microenvironment of the transplant is crucial to know the mechanisms underlying the high early mortality. The study of the microenvironment should take into account the mechanical aspects of transplantation, vascular lesions and the mechanism of in situ coagulation and the supply of nutrients to transplanted cells and the different mechanisms of cell death (apoptosis, autophagy, necrophagy. ).

With regard to the mechanical aspects, the diameter of the needle lumen and the number of cells to be injected should be taken into account. The finer the needle, the greater the pressure exerted on the plunger and the more likely it is to cause lesions on the membrane of these cells to be transplanted or even burst. An important part of the cells can die at this stage without having been introduced into the muscle of the recipient. In this case, both cell debris, damaged cells and whole cells are introduced.

Puncture of the recipient’s muscle with the needle may damage some blood vessels and generate micro-hemorrhages around the transplanted cells. Hemorrhages then activate the mechanisms of vascular and plasma coagulation. It is highly likely that the transplanted cells can be trapped in a fibrin network. This eventuality can be the basis of hypoxia and the lack of nutritional intake that can lead to the death of transplanted cells. One study showed that migration of transplant cells could be improved by using a plasminogen activator, urokinase. Plasminogen converted by urokinase to plasmin can degrade the fibrin formed during coagulation as well as the extracellular matrix of the muscle fibers [41].

One of the mechanisms of cell death that has not yet been studied even partially is autophagy [42]. This death of a number of cells permits the survival of other cells. It is a form of cannibalism at the cellular level. Indeed, the macromolecules of the dead cells following autophagy are degraded by the autophagolysosomal machinery into small molecules that can be assimilated by the surviving cells. All transplantation trials indicated the survival of a small core of transplanted cells that differentiate and fuse with the host muscle fibers [38]. Are these cells that escaped autophagy? Demonstration of enhanced expression of autophagic markers in the micro-environment of the graft may be evidence of activation of this cell death pathway. It can also confirm the lack of nutrients in the microenvironment of the graft.

The Advances and Challenges of Gene Therapy

Before discussing gene therapy, we think a brief reminder of the DMD gene and the protein it encodes, dystrophin, is required for a better understanding of the different gene therapy approaches. The DMD gene is located on the short arm of the X chromosome in the locus 21 (Xp21). It is the largest gene in the human body with 2.4 million base pairs (2.4 Mb). It contains 79 exons (coding sequences) and its complementary DNA (cDNA) is 14 kilobases (14 kb) [5].

The protein encoded by the DMD gene is dystrophin. It is a long filament formed by 3685 amino acids with a molecular weight of 427 kDa. The secondary structure shows that dystrophin consists of 24 spectrin-like repeats (SLRs) and 4 junction regions called hinges (H1, H2, H3 and H4) [43]. Each SLR comprises three alpha helices: helix A, helix B and helix C (Figure 2a and 2b) [44]. Dystrophin allows the junction between a group of proteins in the sarcolemma (the dystrophin associated protein complex, DAPC)) and some cytosol proteins. The dystrophin complex plays a crucial role in maintaining the integrity of sarcolemma during muscle contractions [45]. Dystrophin is a shock absorber at the level of the muscle fiber and its absence, following some mutations, leads to the rupture of the sarcolemma and local necrosis of the muscle cell. 70% of DMD mutations are deletions of one or several exons that lead to a reading frame shift. The other 30% mutations are ponctual mutations that lead to a frame shift or the formation of a stop codon. Most of the deletions occur in the region between exons 44 and 56. This region is considered the hot region of the DMD gene [46,47]. The different approaches of gene therapy that we will describe in the following sections, concern the correction of the DMD gene in this region (Figure 2A,B) [48,49].

Figure 2: Dystrophin complex. A) Dystrophin contains an N-terminus (NT), a filament-shaped central part, a cysteine-rich (CR) domain and a C-terminal (CT). The central part is composed of 24 Spectrin-Like Repeats (SLRs) numbered from 1 to 24 and four junction regions called Hinge numbered H1 to H4. There are two actin binding sites, one of which is located at the NT end and the other at the SLR11-15. SLR1-3 interact with the cell membrane and SLR 16 and 17 with neuronal Nitric Oxide Synthetase (nNOS). Dystrophin also interacts with microtubules at SLR20-23. A portion of H4 and the CR domain bind with the beta-dystroglycan (βDG) protein. CT interacts with syntrophin (Syn) and dystrobrevin (Dbr). Dystrophin complex allows the binding of cytoskeletal proteins (actin and microtubules) with laminin in the extracellular matrix (arrow). Sarcoglycans and sarcospan do not interact directly with dystrophin. However, they stabilize the dystrophin complex. The spectral type repeat α-helices. Image modified from [48]. B) Dystrophin comprises 24 SLRs. Each SLR is formed by 3 alpha helices (helixes A, B and C). The N-terminus (N-ter) is at the beginning of helix A and the C-terminus (C-ter) is at the end of helix C. The helix A is bonded to helix B by loop AB while the helix B is connected to helix C loop BC. Image modified from [49].

Gene therapy aims to develop safe and systemic therapeutic approaches capable of restoring dystrophin expression in all skeletal muscles and heart of Duchenne patients [50]. Given the difficulty in introducing the complete DMD gene into the patient’s muscle fibers due to its large size, delivery of a truncated DMD gene or modifications of the existing DMD gene are being investigated to restore the reading frame either by exon skipping, by exonic knock-out or by the additional deletions of exon fragments. The ultimate aim of these approaches is to produce an internally truncated functional dystrophin protein to transform the Duchenne patient with a severe phenotype into a Becker type patient with a mild phenotype [51]. The idea of producing an internally truncated dystrophin is based on an observation made in a Becker patient who had a 46% deletion of the DMD gene and who lived for about 60 years, walking with a cane [52]. There are also other approaches to introduce a truncated DMD gene that codes either for a mini- or a micro-dystrophin using viral vectors [53,54]. Other approaches aim to suppress a premature stop codon [55].

The advances and challenges of exon skipping

Exon skipping is a gene therapy approach which consists in administering systemic or intramuscular RNA or DNA antisense oligonucleotides (ASO) to the animal model or the patient to mask the splice sites of the exonic sequences to delete them from the final mRNA [56]. With this approach, it is possible to eliminate one or several exons to restore the reading frame and thus allow the translation of an mRNA deleted of a part of its sequence. This permits the expression an internally truncated dystrophin protein which may be more or less functional.

The first experiments of this approach were performed in the mdx mice (a mouse model with a stop codon in exon 23 of the DMD gene) [57] and in myoblasts of patients [58]. Two proof-of-concept clinical trials were conducted by intramuscularly administering 2’-O-methyl-ribo-oligonucleotide-phosphorothioate, Drisapersen (PRO051/GSK402968, Prosensa / Biomarin) [10] and by IV administering phosphorodiamidate morpholino oligomer (PMO) Eteplirsen (AVI 4658/Exondys 51 Sarepta Therapeutics) [59,60] in the patient. These two ASOs generated a specific exon jump of exon 51 and induced the synthesis of the variable amounts of truncated dystrophin.

In case of Drisapersen (GSK402968), the results of the phase III trials did not bring significant improvements of the 6-minute walk test [61]. Several causes may explain these disappointing results: the lack of effect of ASOs in the cardiac muscle, the low penetration of ASOs into the skeletal muscle fibers, and the rapid elimination of ASOs from the circulation, which required repeated administration [50].

This situation led the FDA and the European Union to suspend the treatment of Duchenne muscular dystrophy with Drisapersen. To address this situation, modifications have been made to the exon skip to improve ASO activity. Some authors have used small nuclear RNAs (snRNAs), such as U7snRNAs, inserted into scAAV9 instead of ASOs to generate exon jump [62].

To improve the penetration of PMOs (phosphorodiamidate morpholino oligomers) into muscle fibers, some studies have used CPP (cell penetrating peptide) in complex with the PMOs [59]. To improve bio-distribution, a new class of ASOs has emerged. These are tricyclo-DNA (tcDNA) oligomers. This type of ASO can easily penetrate the skeletal muscles, the heart and the brain [63].

It is in this context that Sarepta Therapeutics initiated Phase I clinical trials in 2014 with PMOs, Eteplirsen (Exondys 51) for skipping of exon 51 that can correct the DMD gene in 13% of Duchenne patients. That clinical trial should have continue until 2019. But unexpectedly, the FDA authorized the commercialization of Eteplirsen as a treatment for DMD. More surprising is the statement made by Mr. Janet Woodcock, director of FDA Center for Drug Evaluation and Research: “Accelerated approval makes this drug available to patients based on initial data, but we eagerly await learning more about the efficacy of this drug through a confirmatory clinical trial that the company must conduct after approval » [64]. This unprecedented and controversial approval was motivated, according to some researchers, by the financial interests and was not based on a scientific basis proving the effectiveness of the drug.

Finally, some authors have proposed to skip multiple exons by administering a mixture of the several ASOs which must target several sequences of the pre-mRNA. Theoretically, it is possible to delete sequences ranging from exon 45 to exon 55 and to hope to correct the DMD gene in 63% of Duchenne patients [65]. However this proposed therapeutic approach does not take into account that exons 42-45 (SLR17) code for a part of the dystrophin protein which binds to nNOS [49]. The absence of nNOS leads to a more severe dystrophy [66-68].

Despite the advances observed in the treatment of DMD by exon skipping, this therapeutic approach has a number of disadvantages. This treatment requires a lifelong administartion of the ASOs because target the mRNA rather than the DMD gene. It is currently difficult to predict the long-term side effects of using ASOs.

Advances and challenges of recombinant endonucleases

In the introduction, we distinguished two types of recombinant endonucleases used for genome editing. These are the endonucleases, which recognize the target sequence by means of a polypeptide chain [12-14], and those, which recognize the target sequence by means of an RNA-type polynucleotide sequence [69]. Both types were used for correction of the dystrophin gene in the myoblasts of Duchenne patients and in different animal models using viral and plasmid vectors [70-73].

Advances and challenges of using meganucleases (MGNs)

Meganucleases are endonucleases that recognize long DNA sequences (14-40 bp) and generate DSBs. They may act as monomers or as homodimers [74-76]. Meganucleases were used to re-establish the reading frame in the Duchenne dystrophic patient’s myoblasts by generating DSBs and micro-insertions or micro-deletions (INDELs) [77]. They were also used in the immortalized myoblasts of the patient to generate DSBs and allow the insertion of exons 45-52. The sequences of these exons (4.5 kb cDNA) were inserted into a donor lentivirus and integrated into the DMD mutated gene of the patient’s myoblasts thus allowing synthesis of a normal dystrophin [71]. Despite its high precision, the production of recombinant meganucleases that can recognize specific DNA sequences remains a challenge and therefore limits their use.

Advances and challenges of Zinc Finger Nucleases (ZFNs)

Zinc Finger Nucleases are hybrid proteins produced from a chimeric gene formed by the fusion of the gene encoding the Zinc Finger Protein (ZFP) [78], on the one hand and the gene encoding the catalytic region of the restriction enzyme FokI on the other hand [13]. ZFPs belong to the Cys2-His2 family of zinc finger proteins. Each zinc finger consists of 25 amino acids of which 10 form the antiparallel β sheet (amino acids 1 to 10) and 12 the α-helix (amino acids 12 to 24). Four amino acids (His 19, His 23, Cys 3 and Cys 6) bind the zinc atom to the α-helix and the β-sheet respectively and stabilize the ZFP structure. The interaction with the DNA takes place at the level of the α-helix. The restriction enzyme FokI is a protein of 587 amino acids produced by the bacterium Flavobacterium okeanokoites. It includes a DNA-binding N-terminus and a catalytic C-terminus [79]. ZFNs act as the left (G) and right (D) antiparallel dimers separated from 5 to 7 nucleotides when linked to DNA [80-82]. Each monomer comprises 3 to 6 zinc finger and each zinc finger recognizes 3 nucleotides. The catalytic portion of FokI is attached to the C-terminus of each monomer. ZFNs were used to correct the mutated DMD gene in mice [83] and in Duchenne cells in culture by excision of exon 51 of the DMD gene [72]. Theoretically, excision of exon 51 can allow the correction of DMD gene in 13% of Duchenne patients. The biggest challenge for the use of ZFNs in gene therapy is the great difficulty in their production.

Advances and challenges of Transcription Activator Like-Effector Nucleases (TALENs)

TALENs are hybrid recombinant proteins produced from a chimeric gene derived from the fusion of the gene encoding the central domain of TALE (Transcription Activator Like-Effector) on one hand and that encoding the catalytic part of FokI [84]. TALEs are proteins produced by Xanthomonas bacteria that infect plants and act as transcription factors. These proteins consist of an N-terminal end with a translocation domain (TD), a DNA binding binding domain and a C-terminal end comprising a localization sequence (NLS) and a transcriptional activation domain (TAD). DNA binding is achieved through the TALE portion. The FokI nuclease is use to induce cleavage. The repeated central domain is formed from 15.5 to 19.5 monomers and each monomer consists of 33 or 34 highly conserved amino acids. The last monomer is composed of only 20 amino acids and is considered as half-monomer. The binding specificity of each monomer with DNA is essentially defined by the amino acid polymorphism at position 12 and 13 (Repeat-Variable Di-residues, RVDs). Each monomer recognizes a nucleotide by the two amino acids located at positions 12 and 13. Thus, the following code is used: HD for attachment to cytosine, NI for adenine, NG for thymine and NN for guanine [85-87]. The repeated central domain can specifically recognize up to 15 nucleotides. Like the ZFNs, TALENS also act as dimers [88]. The monomer G and the monomer D are antiparallel and have, at their C-terminus, the catalytic part of FokI. TALENs corrected the mutated dystrophin gene in myoblasts and fibroblasts [89] and in patient induced pluripotent cells [73]. Like the ZFNs, the challenge of using TALENs as a therapeutic approach against DMD lies in their construction.

Advances and challenges of using the CRISPR/Cas9

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, CRISPR/associated 9 (Cas9) is a defense mechanism used by bacteria to destroy phagic and plasmid DNA [90]. The type most commonly used is type II of Streptococcus pyogenes. When used for genome editing in eukaryotic cells, type II comprises a single guide RNA (sgRNA) sequence formed of two parts: a variable portion of 20 nucleotides, which recognizes the target DNA sequence, and a conserved portion of 110 nucleotides, which stabilizes the binding of the Cas9 nuclease at the cleavage site. For SpCas9 to be recruited on the target sequence, this sequence has to be followed in 3’ by an NGG trinucleotide called Protospacer adjacent motif (PAM) (Figure 3). There are two catalytic sites in Cas9: HNH and RuvC. DSBs are generated exactly at 3 nucleotides from the PAM toward the 5 ‘end of the target DNA [91-95].

Figure 2: The CRISPR / Cas9 system. The DNA target sequence (orange) is illustrated with the two strands separated. The sense strand of target sequence id followed at its 3’ end by the protospacer adjacent motif (PAM), which is NGG (in blue) for the SpCas9 nuclease. The cleavage site (double arrow in red) is located at 3 nucleotides (CGG in mauve) from the PAM towards the 5 ‘end. The variable portion of sgRNA (20 nucleotides in green) recognizes the target sequence by the Watson-Crick base pairing. The nuclease Cas9 (in light gray) is recruited at the PAM. The invariable part of sgRNA (nucleotides in black) stabilizes the whole structure. Image modified from [107].

The main advantage of the CRISPR/Cas9 system over the other endonucleases described above is its production simplicity. The only limiting factor in the targeting of DNA sequences is the presence of the NGG PAM required for the SpCas9 nuclease. However, in recent years, other CRISPR systems (Cpf1, SaCas9, CjCas9. ) [96-98] with different PAMs have been identified in many bacterial species and are being tested, thus increasing our ability to target virtually the entire genome. In addition to ease of production, the CRISPR/Cas9 system has demonstrated high efficiency and accuracy when used in vitro and in vivo in eukaryotic cells and in animal models when inserted into plasmids and viral vectors [99,100]. For Duchenne muscular dystrophy gene therapy, several studies using the CRISPR/Cas9 system have already been published. Some studies aim to produce an internally truncated dystrophin that could be functional by generating intronic DSBs followed by deletions of complete exons to restore the reading frame shift, which was responsible for the presence of a premature stop codon [101-103]. The problem with this approach is that it does not take into account the structure of truncated dystrophin. To function optimally, the structure of the SLR of the truncated dystrophin produced must be correct. Each SLR must be made of a succession of 3 alpha helixes: A, B and C. A disturbance in this arrangement results in poor conformation of the truncated dystrophin and hence in a less optimally functional truncated protein. This explains the existence of numerous Becker phenotypes, some of which (with truncated dystrophins with poor conformation) are severe [104]. It is therefore not enough to restore the reading frame only to produce an internally truncated dystrophin. It is for this reason that we have used the CRISPR-induced deletion method (CinDel) [105,106]. CinDel aims to generate DSBs with two sgRNA and the Cas9 nuclease in the exons preceding and following the patient deletion, to induce additional deletions of portions of the target exons and of intron sequences located between these two DSBs. This generates a hybrid exon, which not only restores the reading frame but also allows the expression of an internally truncated dystrophin. The judicious choice of sgRNAs makes it possible to produce a dystrophin whose structure respects the order of succession of the helices in the SLRs, which may have a good conformation and which will functional optimally. The in vitro tests on the myoblasts of the patient and in vivo on the animal model confirmed the synthesis of an internally truncated dystrophin. Some other authors aim to generate DSBs and insert a short cDNA fragment using a viral vector. By homologous recombination, the dystrophin gene can be corrected and normal dystrophin produced [99]. The CRISPR/Cas9 system has revolutionized the gene therapy approach and to date, numerous studies are being conducted around the world to effectively treat a large number of monogenic genetic diseases. The major challenge in using this approach is the possibility of off-target mutations (Figure 3) [107].

Replacing the DMD mutated gene with a mini-dystrophin synthetic gene

In 2006, Asklepios Biopharmaceuticas Inc. undertook a Phase I clinical trial of its product BiostrophinR, a mini-dystrophin gene sequence consisting of the N-terminal domain (NTD), hinge 1 (H1), SLR1-3), hinge 3 (H3), SLR20-24), hinge 4 (H4), a cysteine rich domain (CRD) and the C terminal domain. In summary, this is a mini-gene having this structure: NTD-H1-R1-R2-R3-H3-R20-R-21-R22-R23-R24-H4-CRD-NTD. It has a size of about 4.5 Kb and was inserted into an AAV2.5 under a CMV promoter. The product was administered intra-muscularly to 6 patients [108]. The test did not improve the condition of the patients and an immune reaction was observed against the transgene and the viral vector [9].

Suppression of the nonsense mutations

About 10% of DMD patients have a nonsense point mutation in the dystrophin gene. This type of mutation leads to premature termination of dystrophin mRNA translation and ultimately to the absence of dystrophin in the patient’s muscle fibers [47]. PTC-Therapeutics Inc. has developed the Atularen R product (PTC-124), a drug, which permits to bypass the premature stop codon and allowing the synthesis of dystrophin in some dystrophic patients. A phase III clinical trial was performed in 174 patients [109]. The results of this trial indicated that this approach could be used to treat some DMD patients [110]. However, the lifetime use of this drug may have undesirable effects.

The model animals of muscular dystrophy

Approaches of gene therapy for DMD invariably follows the following pathway: in vitro test in patient cells or in vivo tests in animal models, clinical trials Phase I, Phase II and Phase III. To date, there are several animal models used to better understand the mechanisms governing the disease and used for preclinical trials [48]. Each model has advantages and disadvantages. In the next section, we will examine some models that are commonly used for DMD.

The mdx mouse contains a nonsense point mutation (a C to T transition) in exon 23 of dystrophin gene [111]. This type of mutation, as mentioned above, represents only about 10% of cases in humans [47]. The use of immortalized DMD cells in vitro does not pose a problem with regard to the type of mutation, however, the use of a model, which has a different phenotype and a different mutation, is not the best situation. The vast majority of preclinical in vivo assays have been performed in the mdx mouse animal model. Compared to the DMD phenotype, the phenotype of the mdx mouse is far less severe. There are many reasons for this difference. First in mdx mice, there is an over-expression of utrophin, a cytosolic protein that has a structure similar to that of dystrophin, which may compensate for the absence of dystrophin [112]. The second reason is the almost continuous renewal of mouse satellite cells (myogenic progenitor cells), which provide a sustained repair of damaged muscle fibers [113]. The third reason is the presence of the cytidine monophosphate sialic acid hydroxylase (CMHA) gene in mdx mice, whereas this gene is repressed in humans [114]. The fourth reason is the small size of the model and therefore its low muscle mass [115]. For all these reasons, some authors claim that the mdx model is not the best and that model results are not often transposable to humans [19].

Instead of mdx mice, it would be better to use hDMD/mdx mice [116]. It is an mdx mouse in which the complete (2.4 Mb) DMD gene has been introduced into the zygote by fusing it with a Yeast Artificial Chromosome (YAC) spheroblast containing the transgene. The human transgene located on chromosome 5 of the mdx mouse can be mutated (our team is already working with a view to producing hDMD/mdx mice with the transgene deleted of some exons using the CRISPR/Cas9 system to reproduce the same mutations as in humans). This new model could then be corrected with the therapy approaches described above. This model has the advantage that the corrections are made on the human gene using the same sgRNAs as used for the DMD patient myoblasts. Thus the results can be, to some extent, transposed to humans. Thus the sgRNA/Cas9, ZFN and TALEN showing activity on the transgene, will also be active on the same gene in humans.

The earliest descriptions of dystrophic dogs date back more than 50 years [117]. Among the different models, Golden Retriever Muscular Dystrophy (GRMD) [118,119] and Cavalier King Charles Spaniel Muscular Dystrophy (CKCS-MD) are the best known [120]. GRMD is the result of a mutation at the splice site at intron 6 (5 ‘splicer donor). This mutation leads to the exon 7 skipping during the maturation of dystrophin mRNA, this causes a premature termination of translation and the absence of dystrophin in the muscle fibers of the dog. In the case of CKCS-MD, this is also a mutation at the splice site in intron 50 (5 ‘splicer donor). This mutation leads to the skipping of exon 50 in the dystrophin mRNA and to a reading frame shift leading to the absence of dystrophin. The canine models have a more severe phenotype and are close to the human phenotype than the mdx mouse model. Unfortunately, this model is very expensive and also difficult to reproduce.

Dystrophic rats were produced by deleting exons 3-16 of the rat DMD gene with two sgRNA/Cas9. The sgRNAs and the Cas9 mRNA were injected into the rat zygotes generating the deletion of the target exons. The joining of the exons 2 and 17 generated a reading frame shift preventing the synthesis of complete dystrophin protein. Besides the increase in size (about 10 times the size of the mdx mouse), mdx rats exhibit a more severe disease phenotype. Hypertrophy of certain skeletal and cardiac muscles is observed [121]. Another model of dystrophic rat produced with TALENs by deleting exon 23, also showed a severe phenotype as the animal advanced in age. The presence of fibrosis and the infiltration of adipose tissue in certain skeletal muscles has been noted [122]. This is model, which is not too expensive, that can be used to replace the mdx mouse.

Dystrophic rhesus macaque

To produce the dystrophic macaques, exons 4 and 46 belonging to two respective hot-spot regions of the DMD gene (exon region 3-7 and exons 44-56 region) were targeted. Both sgRNAs and Cas9 mRNA were injected into rhesus macaque zygotes. These sgRNA/Cas9 generated DSBs and INDELs at the target sites. Some of these INDELs induced a reading frame shift or premature stop codons into the mutated gene [123]. The use of a non-human primate model despite its high cost could be a crucial step before clinical trials.

The dystrophic pig model was produced to effectively solve the problem of low muscle mass in the vast majority of animal models used. This model also showed a phenotype as severe as the human phenotype of DMD [124].

Risks / Benefits

What are the risks and benefits of gene therapy?

With the exception of Luxturna which has been FDA approved, doctors are still experimenting with gene therapy. The long-term safety of such treatments has yet to be determined. Some gene therapies appear to be effective in curing certain conditions. But there is not enough evidence about gene therapy as a whole to determine all the possible risks.

Some gene therapy research indicates gene therapy may worsen symptoms or cause them to last longer. Additionally, complications of certain gene therapies may include cancer, toxicity and inflammation.

A rare but devastating disease

Francis Collins, former leader of the Human Genome Project, had worked on progeria for many years before the breakthrough.

Children carrying the mutation for progeria have normal intelligence but show early signs of general ageing, including hair loss and hearing loss. By their teenage years they appear very old. Few live past the age of 13.

An estimated 400 children around the world live with progeria. The disease’s official name is Hutchinson-Gilford syndrome. Wes Stafford/AP

In 2003, Collins’s lab discovered progeria is caused by a mutation (which you can think of as a “misspelling”) in a gene that encodes a protein called Lamin A. Lamin A has a structural role in the cell’s nucleus.

Many of us carry mutations in various genes. But as we typically have two copies of genes (one from our mother and one from our father), we tend to have at least one good copy and that’s usually enough.

But the progeria mutation in Lamin A is different. While there may be a good copy present, the mutant copy generates a poisonous product that messes things up, like a spanner in the works. This type of mutation is called a “dominant negative mutation”.

The solution, ideally, would be to specifically correct the mutant copy using CRISPR. With this gene-editing tool, scientists can direct a pair of molecular “scissors” to any part of the genome (DNA). Unfortunately, first-generation CRISPR technologies — while good at cutting genes — do not have the level of surgical precision or efficiency needed to correct the Lamin A mutation.

Possible complications due to gene-therapy? - Biology

Measuring the success of treatment is just one challenge of gene therapy. Research is fraught with practical and ethical challenges. As with clinical trials for drugs, the purpose of human gene therapy clinical trials is to determine if the therapy is safe, what dose is effective, how the therapy should be administered, and if the therapy works. Diseases are chosen for research based on the severity of the disorder (the more severe the disorder, the more likely it is that it will be a good candidate for experimentation), the feasibility of treatment, and predicted success of treatment based on animal models. This sounds reasonable. However, imagine you or your child has a serious condition for which no other treatment is available. How objective would your decision be about participating in the research?

How do researchers determine which disorders or traits warrant gene therapy? Unfortunately, the distinction between gene therapy for disease genes and gene therapy to enhance desired traits, such as height or eye color, is not clear-cut. No one would argue that diseases that cause suffering, disability, and, potentially, death are good candidates for gene therapy. However, there is a fine line between what is considered a "disease" (such as the dwarfism disorder achondroplasia) and what is considered a "trait" in an otherwise healthy individual (such as short stature). Even though gene therapy for the correction of potentially socially unacceptable traits, or the enhancement of desirable ones, may improve the quality of life for an individual, some ethicists fear gene therapy for trait enhancement could negatively impact what society considers "normal" and thus promote increased discrimination toward those with the "undesirable" traits. As the function of many genes continue to be discovered, it may become increasingly difficult to define which gene traits are considered to be diseases versus those that should be classified as physical, mental, or psychological traits.

To date, acceptable gene therapy clinical trials involve somatic cell therapies using genes that cause diseases. However, many ethicists worry that, as the feasibility of germ line gene therapy improves and more genes causing different traits are discovered, there could be a "slippery slope" effect in regard to which genes are used in future gene therapy experiments. Specifically, it is feared that the acceptance of germ line gene therapy could lead to the acceptance of gene therapy for genetic enhancement. Public debate about the issues revolving around germ line gene therapy and gene therapy for trait enhancement must continue as science advances to fully appreciate the appropriateness of these newer therapies and to lead to ethical guidelines for advances in gene therapy research. Major participants in the public debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

Risks of genetic engineering

by Anastasia Bodnar 25 September 2012

By Anastasia Bodnar and Karl Haro von Mogel

It seems like every news article about genetic engineering gives a nod to unknown risks to the environment or human health that are unique to genetic engineering. What are those risks, and are they really unique?

Before we get into the details of specific potential risks, there are three things we need to consider.

  1. Is a risk unique to genetic engineering as a whole or risks of individual traits of categories of traits? Each individual trait, whether bred or engineered, must be examined for safety and appropriateness in the situation in which it will be used.
  2. What is the risk compared to alternatives? Risk associated with a genetically engineered trait may be less than the risk associated with a practice that it will replace.
  3. What is the source of the risk? Is it something inherent in the trait or are there external factors?

Gene transfer

One of the most common concerns is with the process of genetic engineering – the transfer of one or more genes from one species to another and potential for unintended genetic changes during the process. While it is possible that unintended changes can occur, this risk is not unique to genetic engineering. Natural and induced mutagenesis as well as traditional breeding methods can also introduce unintended genetic changes, resulting in additional genes being turned on or off, deletions, duplication, and other changes in the genome. Crossing crop plants with wild relatives can introduce genes and proteins that have never been in the human food supply, as can genes inserted through genetic engineering.

Monoculture farming

In the United States and Canada, genetically engineered crops are generally used in farms grown with a monoculture system. However, these farms were monocultures before the advent of genetic engineering and would continue to be monocultures if genetic engineering disappeared tomorrow. Monoculture farming is a problem in and of itself, separate from genetic engineering. In India and Africa, genetically engineered crops (specifically Bt cotton and Bt maize) is grown in a variety of systems. Virus resistant papaya is grown in both larger production farms and in people’s backyards.

Pest resistance

Whenever a pesticide is used year after year in the same place on the same target populations, resistance to that pesticide will develop. This is not a problem specific to genetic engineering. With any pesticide, and with any genetically engineered crop that involves pests, the pesticides must be rotated to prevent any resistance genes that develop from spreading throughout the population. While crops resistant to glyphosate have resulted in a switch away from other herbicides to glyphosate which has resulted in an increase in glyphosate resistant weeds, this is not due to genetic engineering. The problem is a lack of integrated pest management strategies that incorporate a variety of solutions.

Microorganisms and other non target organisms

Another concern that some people have is about genetically engineered crops is that they might have a negative impact on soil microorganisms, beneficial insects, and other wildlife. It is possible that some genetically engineered traits might impact wildlife. Of course, each trait must be assessed individually and we must determine what is the relative risk compared to other options. For example, it is possible that a particular type of Bt in maize would have an effect on soil microorganisms compared to a similar maize without Bt, but that many pesticides against root worms would be even more disruptive for soil microorganisms.

There is one particular example that we need to address in this section. We’ve all heard the story about Monarch butterflies being harmed by genetically engineered crops. First, it was claimed that Bt in pollen harmed the butterflies. Thankfully, those claims were wildly exaggerated – the result of experiments that did not match real-world conditions. Next, it was claimed that monarchs are harmed because the herbicide glyphosate is being used to kill weeds, including the milkweed that Monarchs need to live. However, this is not a problem of glyphosate resistant crops. It is a symptom of a larger problem – that of sterile lawns without weedy flowers that feed butterflies and of farms that are planted border to border without much wild land between.

Modifying biochemical pathways

Plants have enormously complex networks of biochemical pathways that create almost every substance that the plants need. These pathways, like a network of roads and highways, are interconnected and have only begun to be understood. Some of the kinds of genetically engineered crops that are beginning to emerge involve modifying these pathways to produce more of a desired substance or less of an undesired one. For instance, some work on Cassava focuses on reducing the amount of toxic compounds in the roots, and the well-known Golden Rice Project involves boosting beta-carotene in the grain to combat vitamin deficiency. It is possible that modifying the levels of these and other substances in the plant can have effects on other parts of the system, and modify the plant in some undesirable way.

This kind of risk must also be taken in the context of the history of plant breeding. The crops we eat today are replete with examples where drastic changes in biochemical pathways have occurred through simple breeding with no understanding of the underlying biochemical mechanisms. For instance, carrots were not originally orange but white and purple in color. The accumulation of genetic mutations, and the long process of breeding resulted in a nutritionally modified vegetable that is today one of the richest sources of beta-carotene in our diets today. The biochemical pathway that produces beta-carotene in orange carrots and Golden Rice is the same, and the risks involved in modifying such pathways would therefore be similar.

What if it works?

Modifying the nutritional content of foods through genetic engineering has the potential to reduce suffering and improve human welfare. Modifying a plant to resist insects has the potential to reduce insecticide sprays and improve yields and/or food security. There are countless other risks involved in agriculture and food that are dealt with on a regular basis. So when discussing the risks involved in genetic engineering, it is important to consider the risk that it will succeed in reducing or mitigating these other risks.

When evaluating the risk of doing something, you should also consider the risks involved with not doing something. As with driving a car or having electricity in your home, there are benefits that come along with the risks of genetic engineering – and all of these need to be taken into account together.

These are just a few examples of broad claims of risk that are often attributed to genetic engineering that are much more complex issues when you examine them more closely. Can you think of others? Lets hear them in the comments.


Because current therapeutic strategies are only partially successful in curing MPS, novel therapeutic approaches based on GT are being developed for various forms of these diseases. While preclinical studies have already demonstrated the potential benefit of both in-vivo and ex-vivo GT approaches in several MPS mouse models, phase I/II clinical trials are under development for various MPS. These strategies are being implemented with the rationale of providing high levels of the therapeutic enzyme to the patient either by direct infusion of the viral vector or by the engrafted gene-modified HSCs. This will hopefully allow for superior correction also at the level of organs that are more difficult to reach, such as the skeleton and the CNS. The development of strategies that allow us to overcome the BBB obstacle using in-vivo GT approaches with systemic injection is another challenge for the near future. The possibility of directly modifying defective genes by site-specific in-vivo genome editing is also being explored for a number of genetic diseases, including MPS.

The implementation of neonatal screening, which allows for early diagnosis, is of critical importance to ensure timely treatment of congenital disorders such as MPS IH in which a younger age at transplantation together with preservation of cognitive function have been reported as major predictors for superior cognitive development after allogeneic HSCT. Neonatal screening programs have already been developed in the US for SCID and some LSD, and pilot studies are on-going also in Italy in selected regions for ADA-SCID and MPS-IH [18, 65,66,67]. Thanks to the implementation of neonatal screening, and considering that UCB has become the preferential HSC source for allogeneic HSCT in LSD and in particular in MPS IH both in the US and Europe, UCB may also be considered in the future as an alternative source for HSC-GT in newborn/toddlers after dedicated storage of autologous UCB units. In this regard, strategies aimed at the expansion and improvement of transduction of primitive HSCs could expand the field of application of ex-vivo gene therapy [68]. The development of conditioning regimens associated with low extramedullary toxicity, such as those based on monoclonal antibodies selectively depleting blood cells in the BM [17], might open new frontiers to increase the application of HSC-GT at neonatal age.