Treatment of Genetic Disease

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CHAPTER 23 Treatment of Genetic Disease

So little done. So much to do.

ALEXANDER GRAHAM BELL

Many genetic disorders are characterized by progressive disability or chronic ill health for which there is, at present, no effective treatment. Consequently, one of the most exciting aspects of the developments in biotechnology is the prospect of new treatments mediated through gene transfer, RNA modification, or stem cell therapy. It is important, however, to keep a perspective on the limitations of these approaches for the immediate future and to consider, in the first instance, conventional approaches to the treatment of genetic disease.

Conventional Approaches to Treatment of Genetic Disease

Most genetic disorders cannot be cured or even ameliorated using conventional methods of treatment. Sometimes this is because the underlying gene and gene product have not been identified so that there is little, if any, understanding of the basic metabolic or molecular defect. If, however, this is understood then dietary restriction, as in phenylketonuria (p. 167), or hormone replacement, as in congenital adrenal hyperplasia (p. 174), can be used very successfully in the treatment of the disorder. In a few disorders, such as homocystinuria (p. 172) and some of the organic acidurias (p. 183), supplementation with a vitamin or co-enzyme can increase the activity of the defective enzyme with beneficial effect (Table 23.1).

Table 23.1 Examples of Various Methods for Treating Genetic Disease

Treatment	Disorder
Enzyme Induction by Drugs
Phenobarbitone	Congenital non-hemolytic jaundice
Replacement of Deficient Enzyme/Protein
Blood transfusion	SCID resulting from adenosine deaminase deficiency
Bone marrow transplantation	Mucopolysaccharidoses
Enzyme/Protein Preparations
Trypsin	Trypsinogen deficiency
α₁-Antitrypsin	α₁-Antitrypsin deficiency
Cryoprecipitate/factor VIII	Hemophilia A
β-Glucosidase	Gaucher disease
α-Galactosidase	Fabry disease
Replacement of Deficient Vitamin or Coenzyme
B₆	Homocystinuria
B₁₂	Methylmalonic acidemia
Biotin	Propionic acidemia
D	Vitamin D–resistant rickets
Replacement of Deficient Product
Cortisone	Congenital adrenal hyperplasia
Thyroxine	Congenital hypothyroidism
Substrate Restriction in Diet
Amino acids
Phenylalanine	Phenylketonuria
Leucine, isoleucine, valine	Maple syrup urine disease
Carbohydrate
Galactose	Galactosemia
Lipid
Cholesterol	Familial hypercholesterolemia
Protein	Urea cycle disorders
Drug Therapy
Aminocaproic acid	Angioneurotic edema
Dantrolene	Malignant hyperthermia
Cholestyramine	Familial hypercholesterolemia
Pancreatic enzymes	Cystic fibrosis
Penicillamine	Wilson disease, cystinuria
Drug/Dietary Avoidance
Sulfonamides	G6PD deficiency
Barbiturates	Porphyria
Replacement of Diseased Tissue
Kidney transplantation	Adult-onset polycystic kidney disease, Fabry disease
Bone marrow transplantation	X-linked SCID, Wiskott-Aldrich syndrome
Removal of Diseased Tissue
Colectomy	Familial adenomatous polyposis
Splenectomy	Hereditary spherocytosis

SCID, Severe combined immunodeficiency.

Protein/Enzyme Replacement

If a genetic disorder is found to be the result of a deficiency of or an abnormality in a specific enzyme or protein, treatment could, in theory, involve replacement of the deficient or defective enzyme or protein. An obviously successful example of this is the use of factor VIII concentrate in the treatment of hemophilia A (p. 309).

For most of the inborn errors of metabolism in which an enzyme deficiency has been identified, recombinant DNA techniques may be used to biosynthesize the missing or defective gene product; however, injection of the enzyme or protein may not be successful if the metabolic processes involved are carried out within cells and the protein or enzyme is not normally transported into the cell. Modifications in β-glucocerebrosidase as used in the treatment of Gaucher disease enable it to enter the lysosomes, resulting in an effective form of treatment (p. 178). Another example is the modification of adenosine deaminase (ADA) by an inert polymer, polyethylene glycol (PEG), to generate a replacement enzyme that is less immunogenic and has an extended half-life.

In some genetic disorders, drug therapy is possible; for example, statins can help to lower cholesterol levels in familial hypercholesterolemia (p. 175). Statins function indirectly through the low-density lipoprotein (LDL) receptor by inhibiting endogenous cholesterol biosynthesis at the rate-limiting step that is mediated by hydroxymethyl glutaryl co-enzyme A (HMG-CoA) reductase. This leads to upregulation of the LDL receptor and increased LDL clearance from plasma.

In others, avoidance of certain drugs or foods can prevent the manifestation of the disorder, for example sulfonamides in glucose-6-phosphate dehydrogenase (G6PD) deficiency (p. 187). Drug therapy might also be directed at a subset of patients according to their molecular defect. An example is a trial in which gentamicin was administered via nasal drops to patients with cystic fibrosis. Aminoglycoside antibiotics such as gentamicin or PTC124 cause read-through of premature stop codons in vitro and only patients with nonsense mutations (p. 25) showed evidence of expression of full-length cystic fibrosis transmembrane conductance regulator (CFTR) protein in the nasal epithelium. However, although gentamicin and PTC124 were effective in the mdx mouse model of Duchenne muscular dystrophy (DMD), a clinical trial with 174 patients failed to show convincing functional improvement after daily treatment for 48 weeks.

Tissue Transplantation

Replacement of diseased tissue has been a further option since the advent of tissue typing (p. 387). An example is renal transplantation in adult polycystic kidney disease or lung transplantation in patients with cystic fibrosis.

Islet transplantation for treating type 1 diabetes mellitus was transformed in 2000 with development of the ‘Edmonton’ protocol. Islet cells are prepared from donated pancreases (usually two per patient) and injected into the liver of the recipient: at 3 years post-transplant more than 80% of patients are still producing their own insulin.

Therapeutic Applications of Recombinant DNA Technology

The advent of recombinant DNA technology has also led to rapid progress in the availability of biosynthetic gene products for the treatment of certain inherited diseases.

Biosynthesis of Gene Products

Insulin used in the treatment of diabetes mellitus was previously obtained from pig pancreases. This had to be purified for use very carefully, and even then it occasionally produced sensitivity reactions in patients. However, with recombinant DNA technology, microorganisms can be used to synthesize insulin from the human insulin gene. This is inserted, along with appropriate sequences to ensure efficient transcription and translation, into a recombinant DNA vector such as a plasmid and cloned in a microorganism such as Escherichia coli. In this way, large quantities of insulin can be made. An artificial gene that is not identical to the natural gene needs to be constructed for this purpose. However, synthetically produced genes cannot contain the non-coding intervening sequences, or introns (p. 17), found in the majority of structural genes in eukaryotic organisms, as microorganisms such as E. coli do not possess a means for splicing of the messenger RNA (mRNA) after transcription.

Recombinant DNA technology is being employed in the production of a number of other biosynthetic products (Table 23.2). The biosynthesis of medically important peptides in this way is usually more expensive than obtaining the product from conventional sources because of the research and development involved. For example, the cost of treating one patient with Gaucher disease can exceed £100,000 per year. However, biosynthetically derived products have the dual advantages of providing a pure product that is unlikely to induce a sensitivity reaction and one that is free of the risk of chemical or biological contamination. In the past, the use of growth hormone from human cadaver pituitaries has been associated with the transmission of Creutzfeldt-Jakob disease, and human immunodeficiency virus (HIV) has been a contaminant in cryoprecipitate containing factor VIII used in the treatment of hemophilia A (p. 309).

Table 23.2 Proteins Produced Biosynthetically Using Recombinant DNA Technology

Protein	Disease
Insulin	Diabetes mellitus
Growth hormone	Short stature resulting from growth hormone deficiency
Factor VIII	Hemophilia A
Factor IX	Hemophilia B
Erythropoietin	Anemia
α-Galactosidase A	Fabry disease (X-linked lysosomal storage disorder)
β-Interferon	Multiple sclerosis

Gene Therapy

Gene therapy has been defined by the UK Gene Therapy Advisory Committee (GTAC) as ‘the deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic, or diagnostic purposes’. It includes techniques for delivering synthetic or recombinant nucleic acids into humans; genetically modified biological vectors (such as viruses or plasmids), genetically modified stem cells, oncolytic viruses, nucleic acids associated with delivery vehicles, naked nucleic acids, antisense techniques (e.g., gene silencing, gene correction or gene modification), genetic vaccines, DNA or RNA technologies such as RNA interference, and xenotransplantation of animal cells (but not solid organs).

Advances in molecular biology leading to the identification of many important human disease genes and their protein products have raised the prospect of gene therapy for many genetic and non-genetic disorders. The first human gene therapy trial began in 1990, but it is important to emphasize that, although it is often presented as the new panacea in medicine, progress to date has been limited, and there are many practical difficulties to overcome before gene therapy can deliver its promise.

Regulatory Requirements

There has been much publicity about the potential uses and abuses of gene therapy. Regulatory bodies have been established in several countries to oversee the technical, therapeutic, and safety aspects of gene therapy programs (p. 368). There is universal agreement that germline gene therapy, in which genetic changes could be distributed to both somatic and germ cells, and thereby be transmitted to future generations, is morally and ethically unacceptable. Therefore all programs are focusing only on somatic cell gene therapy, in which the alteration in genetic information is targeted to specific cells, tissues or organs in which the disorder is manifest.

In the USA the Human Gene Therapy Subcommittee of the National Institutes of Health has produced guidelines for protocols of trials of gene therapy that must be submitted for approval to both the Food and Drug Administration and the Recombinant DNA Advisory Committee, along with their institutional review boards. In the United Kingdom, the GTAC provides ethical oversight of proposals to conduct clinical trials involving gene or stem cell therapies in humans, taking account of the scientific merits, and the potential benefits and risks.

More than 1500 clinical trials of gene therapy have been approved for children and adults for a variety of genetic and non-genetic disorders. For the most part, these appear to be proceeding without event, although the unexpected death of a patient in one trial in 1999 and the development of leukemia in 3 of 20 children who received gene therapy for X-linked severe combined immunodeficiency (XL-SCID) (p. 201) has highlighted the risks of gene therapy.

Technical Aspects

Before a gene therapy trial is possible, there are a number of technical aspects that must be addressed.

Animal Models

One of the basic prerequisites for assessing the suitability of gene therapy trials in humans is the existence of an animal model. Although there are naturally occurring animal models for some inherited human diseases, for most there is no animal counterpart. The techniques used to generate animal models for human disease are outside the scope of this book, but much effort has focused on the production of animal models that faithfully recreate disease phenotypes. Animal models for cystic fibrosis, DMD, Huntington disease, and Friedreich ataxia have been generated and provide just a few examples that may be used to evaluate gene therapy before trials in humans.

In Utero Fetal Gene Therapy

The report of successful adenovirus vector-mediated in utero gene therapy in a cystic fibrosis mouse model in 1997 means that fetal gene therapy in utero may be possible in humans. At present it is considered unacceptable because of the possibility of inadvertent germ-cell modification. The use of stem cells genetically modified ex vivo should reduce this risk. However, in utero stem-cell transplantation without genetic modification offers the best prospects for the successful treatment of serious neurodegenerative disorders with a very early onset, such as Krabbe disease or Hurler syndrome (p. 177).

Gene Transfer

Gene transfer can be carried out either ex vivo by treatment of cells or tissue from an affected individual in culture, with reintroduction into the affected individual, or in vivo if cells cannot be cultured or be replaced in the affected individual (Figure 23.1). The ex vivo approach is limited to disorders in which the relevant cell population can be removed from the affected individual, modified genetically, and then replaced. The in vivo approach is the most direct strategy for gene transfer and can theoretically be used to treat many hereditary disorders.

FIGURE 23.1 In vivo and ex vivo gene therapy. In vivo gene therapy delivers genetically modified cells directly to the patient. An example is CFTR gene therapy using liposomes or adenovirus via nasal sprays. Ex vivo gene therapy removes cells from the patient, modifies them in vitro and then returns them to the patient. An example is the treatment of fibroblasts from patients with hemophilia B by the addition of the factor IX gene. Modified fibroblasts are then injected into the stomach cavity.

Target Organs

In many instances, gene therapy will need to be, and should be, directed or limited to a particular organ, tissue or body system.

Liver

Liver cells are susceptible to transfection by retroviruses in vitro. Cells removed from the liver by partial hepatectomy can be treated in vitro and then reinjected via the portal venous system, from which they seed in the liver. Hypercholesterolemia is a major cause of cardiovascular disease in the Western world. The most severe form, autosomal recessive familial hypercholesterolemia, is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Patients do not respond to statin therapy, require maintenance therapy with highly invasive LDL apheresis, and typically die of myocardial infarction in their third decade of life. Gene therapy for lipid disorders has a high potential for success, but studies to date based on viral-mediated overexpression of LDLR cDNA have been unsuccessful, probably because vectors have lacked the sterol response elements that are required for regulated transcription.

Central Nervous System

Gene therapy for central nervous system disorders, such as Parkinson and Alzheimer diseases, requires delivery to the brain. Lentiviral vectors are particularly suitable because they integrate into the host genome of non-dividing cells and can potentially act as a delivery system for stable expression. After a study in a non-human primate model of Parkinson disease that reported motor deficit improvements following the use of a lentiviral vector to restore extracellular dopamine levels, several clinical trials have been undertaken to evaluate the effectiveness of this approach in humans. Encouraging results came from a study in 2009 in which the neurotransmitter GABA was delivered to one side of the brain in 12 patients with Parkinson disease and improvement demonstrated by positron emission tomography scanning. Trials for Alzheimer disease are at an earlier stage, but results of a phase II trial of 50 patients with Parkinson disease to deliver nerve growth factor into the brain are eagerly awaited.

Muscle

Unlike other tissues, direct injection of foreign DNA into muscle has met with some success in terms of retention and expression of the foreign gene in the treated muscle. However, this will not provide a long-term response. A clinical trial is underway to introduce a ‘microdystrophin’ molecule, so called because it contains only the bare essential domains of the dystrophin gene, via an adeno-associated viral (AAV) vector into the arm muscles of patients with DMD.

Bone Marrow

In the treatment of disorders affecting the bone marrow, problems arise from the small numbers of stem cells, which need to be immortalized. Ex vivo gene therapy has been reported in two young boys with X-linked adrenoleukodystrophy for whom suitable bone marrow donors were not available. Stem cells were removed from their bone marrow, genetically corrected with a lentiviral vector encoding the normal ABCD1 gene, and then re-infused after the patients received myeloablative treatment. A year later, the progressive cerebral demyelination had stopped, a clinical outcome comparable to that achieved by allogeneic bone marrow transplant.

There are two main methods for delivering gene transfer, viral and non-viral.

Viral Agents

A number of different viruses can be used to transport foreign genetic material into cells and the most successful viral agents are described in the following sections.

Lentiviruses

The lentivirus family includes HIV. Lentiviruses are complex viruses that infect macrophages and lymphocytes, but their main advantage is that they can be integrated into non-dividing cells. They may, therefore, be useful in the treatment of neurological conditions.

Adenoviruses

Adenoviruses can be used as vectors in gene therapy as they infect a wide variety of cell types. They are stable, can infect non-dividing cells and carry up to 36 kb of foreign DNA. In addition, they are suitable for targeted treatment of specific tissues such as the respiratory tract, and have been extensively used in gene therapy trials for the treatment of cystic fibrosis.

Adenoviruses do not integrate into the host genome, thereby avoiding the possibility of insertional mutagenesis but having the disadvantage that expression of the introduced gene is usually unstable and often transient. They also contain genes known to be involved in the process of malignant transformation, so there is a potential risk that they could inadvertently induce malignancy. By virtue of their infectivity, they can produce adverse effects secondary to infection and by stimulating the host immune response. This was demonstrated by a vector-related death following intravascular administration of high doses (3.8 × 10¹³) of adenovirus particles to a patient with ornithine transcarbamylase deficiency.

Adeno-Associated viruses

Adeno-associated viruses are non-pathogenic parvoviruses in humans that require co-infection with helper adenoviruses or certain members of the herpes virus family to achieve infection. In the absence of the helper virus, the adeno-associated virus DNA integrates into chromosomal DNA at a specific site on the long arm of chromosome 19 (19q13.3-qter). Subsequent infection with an adenovirus activates the integrated adeno-associated viral DNA-producing virions. They have the advantages of being able to infect a wide variety of cell types, exhibiting long-term gene expression and not generating an immune response to transduced cells. The safety of adeno-associated viruses as vectors occurs by virtue of their site-specific integration but, unfortunately, this is often impaired with the inclusion of foreign DNA in the virus. The disadvantages of adeno-associated viruses include the fact that they can be activated by any adenovirus infection and that, although 95% of the vector genome is removed, they can take inserts of foreign DNA of only up to 5 kb in size.

Non-Viral Methods

There are a number of different non-viral methods of gene therapy but the most popular is liposome-mediated DNA transfer. This has the theoretical advantage of not eliciting an immune response, being safer and simpler to use as well as allowing large-scale production, but efficacy is limited.

Liposomes

Liposomes are lipid bilayers surrounding an aqueous vesicle that can facilitate the introduction of foreign DNA into a target cell (Figure 23.2). A disadvantage of liposomes is that they are not very efficient in gene transfer and the expression of the foreign gene is transient, so that the treatment has to be repeated. An advantage of liposome-mediated gene transfer is that a much larger DNA sequence can be introduced into the target cells or tissues than with viral vector systems.

FIGURE 23.2 Diagrammatic representation of liposome-mediated gene therapy.

RNA Modification

RNA modification therapy targets mRNA, either by suppressing mRNA levels or by correcting/adding function to the mRNA.

Antisense Oligonucleotides

Antisense therapy may be used to modulate the expression of genes associated with malignancies and other genetic disorders. The principle of antisense technology is the sequence-specific binding of an antisense oligonucleotide (typically 18 to 30 bases in length) to a target mRNA that results in inhibition of gene expression at the protein level.

The identification of exon-splicing enhancer (ESE) sequences has increased our understanding of the process of exon splicing. If an ESE is mutated, the exon is more likely to be spliced out. Some proteins with in-frame whole-exon deletions retain some residual activity; for example, dystrophin mutations in Becker muscular dystrophy (p. 307). Blocking an ESE using an antisense oligonucleotide in patients with DMD aims to restore the reading frame and ameliorate the phenotype to that of Becker muscular dystrophy. However, a specific antisense oligonucleotide is required to target each different exon. The potential for antisense therapy is perhaps greater in spinal muscular atrophy (p. 306) where the non-expressed SMN2 gene could be converted to generate functional SMN1 protein in virtually all patients.

RNA Interference

This technique also has broad therapeutic application, as any gene may be a potential target for silencing by RNA interference. In contrast to antisense oligonucleotide therapy where the target mRNA is bound, as a result of RNA interference the target mRNA is cleaved and it is estimated to be up to 1000-fold more active. RNA interference works through the targeted degradation of mRNAs containing homologous sequences to synthetic double-stranded RNA molecules known as small interfering RNAs (siRNAs) (Figure 23.3). The siRNAs may be delivered in drug form using strategies developed to stabilize antisense oligonucleotides, or from plasmids or viral vectors. The first of these drugs is bevasiranib, an siRNA therapy designed to silence the genes that produce vascular endothelial growth factor, believed to be responsible for the vision loss in patients with the “wet” form of age-related macular degeneration.

FIGURE 23.3 Mechanism of RNA interference. Double-stranded (ds) RNAs are processed by Dicer, in an ATP-dependent process, to produce small interfering RNAs (siRNA) of about 21–23 nucleotides in length with two-nucleotide overhangs at each end. Short hairpin (sh) RNAs, either produced endogenously or expressed from viral vectors, are also processed by Dicer into siRNA. An ATP-dependent helicase is required to unwind the dsRNA, allowing one strand to bind to the RNA-induced silencing complex (RISC). Binding of the antisense RNA strand activates the RISC to cleave mRNAs containing a homologous sequence.

(From Lieberman, et al 2003 Trends Mol Med 9:397–403, with permission.)

Targeted Gene Correction

A promising new approach is to repair genes in situ through the cellular DNA repair machinery (p. 27). Proof of principle has been demonstrated in an animal model of Pompe disease. The point mutation was targeted by chimeric double-stranded DNA-RNA oligonucleotides containing the correct nucleotide sequence. Repair was demonstrated at the DNA level and normal enzyme activity was restored.

The latest strategy uses engineered zinc-finger nucleases (ZFNs) to stimulate homologous recombination. Targeted cleavage of DNA is achieved by zinc-finger proteins designed to recognize unique chromosomal sites and fused to the non-specific DNA cleavage domain of a restriction enzyme. A double-strand break induced by the resulting ZFNs can create specific changes in the genome by stimulating homology-directed DNA repair between the locus of interest and an extrachromosomal molecule. One possible application is to manufacture HIV resistant T cells through disruption of the HIV receptor CCR5 by zinc-finger nucleases.

Diseases Suitable for Treatment Using Gene Therapy

Disorders that are possible candidates for gene therapy include both genetic and non-genetic diseases (Table 23.3).

Table 23.3 Diseases that Can Potentially Be Treated by Gene Therapy

Disorder	Defect
Immune deficiency	Adenosine deaminase deficiency Purine nucleoside phosphorylase deficiency Chronic granulomatous disease
Hypercholesterolemia	Low-density lipoprotein receptor abnormalities
Hemophilia	Factor VIII deficiency (A) Factor IX deficiency (B)
Gaucher disease	Glucocerebrosidase deficiency
Mucopolysaccharidosis VII	β-Glucuronidase deficiency
Emphysema	α₁-Antitrypsin deficiency
Cystic fibrosis	CFTR mutations
Phenylketonuria	Phenylalanine hydroxylase deficiency
Hyperammonemia	Ornithine transcarbamylase deficiency
Citrullinemia	Argininosuccinate synthetase deficiency
Muscular dystrophy	Dystrophin mutations
Spinal muscular atrophy	SMN1 gene deletion
Thalassemia/sickle cell anemia	α- and β-globin mutations
Malignant melanoma
Ovarian cancer
Brain tumors
Neuroblastoma
Renal cancer
Lung cancer
AIDS
Cardiovascular diseases
Rheumatoid arthritis

Genetic Disorders

There are a number of single-gene diseases that have been the focus of gene therapy attempts.

Adenosine deaminase deficiency

One of the first diseases for which gene therapy was attempted in humans is the inherited immunodeficiency disorder caused by adenosine deaminase (ADA) deficiency (p. 179). The most successful conventional treatment for ADA deficiency is bone marrow transplantation but, in the absence of a compatible donor, patients may be treated with PEG-conjugated ADA.

In 1990 the first gene therapy trial enrolled 10 patients with ADA-SCID. Although no adverse events were reported, none of the patients was ‘cured’, probably because of the low efficiency of gene transfer from the retroviral vector. More recently, an improved gene transfer protocol in bone-marrow CD34+ cells combined with low-dose chemotherapy resulted in multi-lineage, stable engraftment of transduced progenitors at substantial levels, restoration of immune functions, correction of the ADA metabolic defect, and proven clinical benefit, in the absence of PEG-ADA.

Hemoglobinopathies

Attempts at treating β-thalassemia and sickle-cell disease by gene therapy have not been effective as yet, primarily because the numbers of α- and β-globin chains must be equal (p. 157). Gene therapy must, therefore, be dose-specific, and this is not possible at the present time.

Cystic fibrosis

In contrast to the hemoglobinopathies, cystic fibrosis should be more amenable to gene therapy as the level of functional protein sufficient to produce a clinical response may be as low as 5% to 10% and the lung is a relatively accessible tissue.

However, progress to date has been slow and, although gene therapy can correct the primary and secondary defects associated with cystic fibrosis, the extent and duration of gene expression has been inadequate, owing to the rapid turnover of lung epithelial cells. There have also been concerns about the safety of some current delivery systems, especially following the adenovirus-triggered death of one patient. For viral vectors, the main challenges are access to target cells and host immunity, which prevents efficient readministration. Liposomes have been used in animal models and clinical trials but are also relatively inefficient.

Hemophilia A and B

Hemophilia A and B were thought to be excellent candidates for gene therapy as a modest increase in the level of factor VIII or IX, respectively, will be of major clinical benefit. Trials have used direct intramuscular injection of adeno-associated virus expressing factor VIII or ex-vivo treatment of fibroblasts with plasmid-borne factor IX followed by injection into the stomach cavity. Although initial results were encouraging, the transient rise in factor VIII levels was modest (0.5% to 4% of normal) and these clinical trials were halted. A new way of targeting gene therapy to mouse liver sinusoidal endothelial cells (the cells that are the main source of factor VIII) has been developed in which nanoparticles are coated with hyaluron so that they target these cells. Even 50 weeks after hemophilia A mice were injected with these nanoparticles, levels of factor VIII in the blood were the same as in the blood of normal mice and bleeding times were also similar to those of normal mice.

Duchenne muscular dystrophy

The main difficulty with gene therapy for DMD is the sheer size of the dystrophin gene—the complementary DNA (cDNA) is 14 kb. Current trials use a ‘microdystrophin’ molecule that includes just the bare essential domains within an adeno-associated vector. The vector is delivered by multiple injections into the arm muscle.

An alternative strategy is to use antisense oligonucleotides to force exon skipping and convert out-of-frame deletions that cause DMD to in-frame deletions usually associated with the milder Becker muscular dystrophy phenotype. This approach could be successful for up to 80% of patients with DMD. Proof of concept has been demonstrated in cultured patient cells and the mdx mouse model where dystrophin re-expression was demonstrated. The first clinical trial involved four patients who underwent intramuscular injection of an antisense oligonucleotide to target exon 51. Dystrophin was restored in the vast majority of muscle fibres at levels between 17% and 35%, without any adverse effects. However, intramuscular injection of individual muscles is not feasible as a treatment and various chemistries are under investigation in order to deliver antisense oligonucleotides to muscles throughout the body.

One key hurdle in the use of antisense oligonucleotide therapy is the fact that each different antisense is considered a new drug and requires separate regulatory approval. This makes their development more expensive and not feasible for low prevalence mutations for which there would be insufficient patients for clinical trials.

There is also the possibility of upregulating a dystrophin homolog, utrophin. Immune rejection is not a problem and studies in the mdx mouse have shown significant improvement in muscle function. Pharmacological compounds that enhance utrophin expression in animal models and cultured patient cells have been identified and clinical trials will start shortly.

Leber’s congenital amaurosis

This autosomal recessive disorder is caused by mutations in the RPE65 gene and characterized by poor vision at birth with complete loss of vision in early adulthood. Ten years ago, studies in a naturally occurring dog model (the Briard dog) showed that gene therapy by means of a single operation involving sub-retinal injection of an adeno-associated vector carrying the full length RPE65 gene sequence was both safe and effective. Recent studies in a small number of young adults have replicated these findings and there is much excitement regarding the potential benefits for younger children whose loss of vision is not so far advanced. One obvious advantage for measuring the success of gene therapy in this condition is that a single eye can be treated whilst the other eye serves as a control. This research provides proof of principle that genetic forms of blindness may be reversed.

Stem Cell Therapy

Stem cells are unspecialized cells that are defined by their capacity for self-renewal and the ability to differentiate into specialized cells along many lineages. Embryonic stem cells are pluripotent, which means they can give rise to derivatives of all three germ layers (i.e., all cell types that are found in the adult organism). Somatic stem cells can only differentiate into the cell types found in the tissue from which they are derived (Figure 23.4), but can be isolated from any human, whatever their age. Nowadays the term induced pluripotent stem cell (iPS) is used rather than somatic or adult stem cell.

FIGURE 23.4 Generation of embryonic and somatic stem cells. The fusion of the sperm and egg during fertilization establishes a diploid zygote that divides to create the blastocyst. Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst. ESCs in culture are capable of self-renewal without differentiation and are able to differentiate into all cell types of the endoderm, mesoderm, and ectoderm lineages using appropriate signals. Somatic stem cells are also capable of self-renewal and, with appropriate signals, differentiate into various cell types from the tissue from which they are derived.

Bone-marrow transplantation is a form of somatic stem cell therapy that has been used for more than 40 years. During the past 5 years, cord blood stem cells have emerged as an alternative source. Although these transplants can be an effective treatment for a number of genetic disorders, including ADA deficiency, SCID,X-linked adrenoleukodystrophy, lysosomal storage diseases and Fanconi anemia, the associated risks of infection due to immunosuppression and graft-versus-host disease are high. The main limitation is the lack of a suitable bone-marrow donor or availability of matched cord blood stem cells.

Transplantation of stem cells (e.g., pluripotent hematopoietic stem cells) in utero offers the prospect of a novel mode of treatment for genetic disorders with a congenital onset. The immaturity of the fetal immune system means that the fetus will be tolerant of foreign cells so that there is no need to match the donor cells with those of the fetus. A small number of trials for have been performed but engraftment has so far only been successful in cases of SCID.

Embryonic Stem Cell Therapy

Teratomas (benign) and teratocarcinomas (malignant) are tumors that are found most commonly in the gonads. Their name is derived from the Greek word ‘teratos’ (monster); it describes their appearance well, as these tumors contain teeth, pieces of bone, muscles, skin, and hair. A key experiment demonstrated that if a single cell is removed from one of these tumors and injected intraperitoneally, it acts as a stem cell by producing all the cell types found in a teratocarcinoma.

Mouse embryonic stem cells were first isolated and cultured 25 years ago. Studies of human embryonic stem cells have lagged behind, but the pace of research increased exponentially following the achievement in 1998 of the first cultured human embryonic stem cells.

Embryonic Stem Cells for Transplantation

The ability of an embryonic stem cell (ESC) to differentiate into any type of cell means that the potential applications of ESC therapy are vast. One approach involves the differentiation of ESCs in vitro to provide specialized cells for transplantation. For example, it is possible to culture mouse ESCs to generate dopamine-producing neurons. When these neural cells were transplanted into a mouse model for Parkinson disease, the dopamine-producing neurons showed long-term survival and ultimately corrected the phenotype. This ‘therapeutic cloning’ strategy has been proposed as a future therapy for other brain disorders such as stroke and neurodegenerative diseases. However, after many encouraging small studies of fetal cell transplantation for Parkinson disease, three randomized, double-blind, placebo-controlled studies found no net benefit. Also, patients in two of the studies developed dyskinesias that persisted despite reductions in medication. Further research is needed to understand and overcome the dual problems of unpredictable benefit and troublesome dyskinesias after dopaminergic cell transplantation. In addition, post mortem analysis of patients who received fetal brain cell transplantation revealed that implanted cells are prone to degeneration just like endogenous neurons in the same pathological area, indicating that long-term efficacy of cell therapy of Parkinson disease needs to overcome the degenerating environment in the brain.

Gene Therapy Using Embryonic Stem Cells

An alternate strategy is to use ESCs as delivery vehicles for genes that mediate phenotype correction through gene-transfer technology. One potential barrier to using human ESCs to treat genetic disorders is immunorejection of the transplanted cells by the host. This obstacle might be overcome by using gene transfer with the relevant normal gene to autologous cells (such as cultured skin fibroblasts), transfer of the corrected nucleus to an enucleated egg from an unrelated donor, development of ‘corrected’ ESCs and, finally, differentiation and transplantation of the corrected relevant cells to the same patient (Figure 23.5).

FIGURE 23.5 Embryonic stem cells for gene therapy. The strategy depicted starts with removing cells (e.g., fibroblasts) from a patient with a monogenic disorder and then transferring the normal gene using a vector (or perhaps by correcting the mutation in vitro). The nucleus from a corrected cell is then transferred to an enucleated egg obtained from an unrelated donor by somatic cell nuclear transfer. The egg, now containing the genetically corrected genome of the patient, is activated to develop into a blastocyst in vitro, and corrected autologous stem cells are derived from the inner cell mass. The stem cells are then directed to differentiate into a specific cell type and transferred to the patient, thereby correcting the disorder.

A crucial component of future clinical applications of this strategy is the ability to derive ‘personalized’ human ESC lines using the nuclear transfer technique. Although research on this technology has been controversial, the efficient transfer of somatic cell nuclei to enucleated oocytes from unrelated donors, and the subsequent derivation of human ESC lines from the resulting blastocysts, is a technical hurdle that has recently been overcome.

There has been much debate around the ethical issues of using ESCs and it seems that embryonic stem cells may not be an essential prerequisite, as iPS cells have been found in many more tissues than was once thought possible. Hence iPS cells might be used be used for transplantation.

Induced Pluripotent Stem Cell Therapy

Certain kinds of somatic stem cell seem to have the ability to differentiate into a number of different cell types, given the right conditions. Recent progress in stem cell biology has shown that iPS-derived cells can be used to successfully treat rodent Parkinson disease models, thus solving the problem of immunorejection and paving the way for future autologous transplantations for treating this disease and others.

Mesenchymal Stem Cells

Mesenchymal stem cell (MSC) therapy, through its promise of repair and regeneration of cardiac tissue, represents an exciting avenue of treatment for a range of cardiovascular diseases. Cardiovascular disease is the leading cause of death in developed countries. Although cardiomyocytes retain limited plasticity following maturation, the heart is grossly unable to recover from structural damage.

MSCs are relatively immunopriviledged, lacking both major histocompatibility II and T cell co-stimulatory signal expression, and possess the unique ability to home into sites of myocardial damage when delivered systemically. They are obtained either from the bone marrow of healthy adult volunteers or from the patients themselves, and cultured in vitro with appropriate factors before being delivered to the damaged heart. Animal studies have shown therapeutic benefit via several distinct mechanisms, the most important of which appears to be the abundant secretion of paracrine factors that promote local regeneration. Phase I clinical trials have shown that this approach is safe and the results of phase II trials are eagerly awaited to see if there will be clear clinical benefit.

The genetic disorder retinitis pigmentosa (p. 182) results in the loss of photoreceptors, leading to visual symptoms in the teens and blindness by 40 to 50 years of age. Recently, systemic administration of pluripotent bone marrow–derived MSCs in a rat model has demonstrated improved visual function. This is a potentially exciting development for the future treatment of other forms of retinal degeneration and other ocular vascular diseases such as diabetic retinopathy.

A third application of MSC therapy is in bone repair and metabolic bone diseases such as osteogenesis imperfecta (p. 121) and hypophosphatasia, because MSCs can also differentiate to form bone and cartilage.