The Treatment of Genetic Disease-B�

Chapter 8

Lecture structure

• Modulation of Gene Expression
• Modification of the Somatic Genome by Transplantation
• Gene therapy
• Precision medicine: the present and future of the treatment of mendelian disease

• Increasing Gene Expression from the Wild Type or Mutant Locus

• Therapeutic effects can be obtained by increasing the amount of messenger RNA (mRNA) transcribed from the wild-type locus associated with a dominant disease or from the mutant locus, if the mutant protein retains some function.
• An effective therapy of this type is used to manage hereditary angioedema, a rare but potentially fatal autosomal dominant condition due to mutations in the gene encoding the complement 1 (C1) esterase inhibitor.

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Modulation of Gene Expression

• Affected individuals are subject to unpredictable episodes, of widely varying severity, of submucosal and subcutaneous edema.
• Hereditary angioedema (HAE) is caused by the poor functioning or lack of a protein called C1 inhibitor that is present in your blood and helps control inflammation (swelling) and parts of the immune system. 

• Attacks that involve the upper respiratory tract can be fatal.
• Because of the rapid and unpredictable nature of the attacks, long-term prophylaxis with attenuated androgens, particularly danazol, is often employed.
• Danazol significantly increases the abundance of the C1 esterase inhibitor mRNA by modulating transcription of the gene, presumably from both the normal and mutant loci.
• In the great majority of patients, the frequency of serious attacks is dramatically reduced, although long-term androgen administration is not free of side effects.
• BERINERT is an injectable medicine used to treat swelling and/or painful HAE attacks in adults and children with Hereditary angioedema (HAE). BERINERT contains C1 esterase inhibitor, a protein .

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Treatment by Modifcation of the Genome or its Expression �

cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; Hb F, fetal hemoglobin; HLA, human leukocyte antigen.

• Increasing Gene Expression from a Locus Not Affected by the Disease

• A related therapeutic strategy is to increase the expression of a normal gene that compensates for the effect of mutation at another locus.
• This approach is promising in the management of sickle cell disease and β-thalassemia, for which drugs that induce DNA hypomethylation are being used to increase the abundance of fetal hemoglobin (Hb F), which normally constitutes <1% of total hemoglobin in adults.
• Sickle cell disease causes illness because of both the anemia and the sickling of red blood cells; the increase in the level of Hb F (α₂γ₂) benefits these patients because Hb F is an adequate oxygen carrier in postnatal life and because the polymerization of deoxyhemoglobin S is inhibited by Hb F.
• In β-thalassemia, Hb F restores the imbalance between α and non–α-globin chains, substituting Hb F (α₂γ₂) for Hb A (α₂β₂).

• The normal postnatal decrease in the expression of the γ-globin gene is at least partly due to methylation of CpG residues in the promoter region of the gene.

• Both patients with sickle cell anemia and patients with some forms of β-thalassemia treated with decitabine uniformly display increases in Hb F to levels that are likely to have a significant positive impact on morbidity and mortality.

• Methylation of the promoter is inhibited if a cytidine analogue such as decitabine (5-aza- 2′-deoxycytidine) is incorporated into DNA instead of cytidine.

• The inhibition of methylation is associated with substantial increases in γ-globin gene expression and, accordingly, in the proportion of Hb F in blood.

• The use of inhibitors of γ-globin gene methylation is evolving rapidly, and more effective inhibitors of methylation, with fewer side effects, are likely to be developed.
• The BCL11A protein, is a trans-acting effector of hemoglobin switching that turns off γ-globin production postnatally but nevertheless allows β-globin gene expression.
• Genome editing in hematopoietic stem cells (HSCs) is currently being explored as a method to delete an erythroid enhancer of the BCL11A gene, thereby blocking its expression in the erythroid cell lineage.
• As a result, hemoglobin switching from Hb F to Hb A would not occur, and patients would retain Hb F instead of a hemoglobin containing a mutant β-thalassemia or sickle cell allele.

• Reducing the Expression of a Dominant Mutant Gene Product: Small Interfering RNAs

• The pathology of some inherited diseases results from the presence of a mutant protein that is toxic to the cell, as seen with proteins with expanded polyglutamine tracts, as in Huntington disease, or with disorders such as the inherited amyloidoses.
• The autosomal dominant disorder transthyretin amyloidosis is the result of any of more than 100 missense mutations in transthyretin, a protein produced mainly in liver, that transports retinol (one form of vitamin A) and thyroxine in body fluids.
• The major phenotypes are amyloidotic polyneuropathy, due to deposition of the amyloid in peripheral nerves (causing intractable peripheral sensory neuropathy and autonomic neuropathy), and amyloidotic cardiomyopathy, due to its deposition in the heart.
• Both disorders greatly shorten the life span, and the only current treatment is hepatic transplantation.

• A promising therapy, however, is provided by a technology called RNA interference (RNAi), which can mediate the degradation of a specific target RNA, such as that encoding transthyretin.
• Briefly, short RNAs that correspond to specific sequences of the targeted RNA—termed small interfering RNAs (siRNAs)—are introduced into cells by, for example, lipid nanoparticles or viral vectors.
• Strands of the interfering RNA, approximately 21 nucleotides long, bind to the target RNA and initiate its cleavage, where elimination of the mutant gene product is the goal

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• Patisiran (Onpattro®) is the first FDA approved siRNA for hereditary trasthyretin amyloidosis.

• Induction of Exon Skipping

• Exon skipping refers to the use of molecular interventions to exclude an exon from a pre-mRNA that encodes a reading frame–disrupting mutation, thereby rescuing expression of the mutant gene.
• If the number of nucleotides in the included exons is a multiple of three, no frame shift will occur and, if the resulting polypeptide with the deleted amino acids retains sufficient function, a therapeutic benefit will result.
• The most widely studied method of inducing exon skipping is through the use of antisense oligonucleotides (ASOs), which are synthetic 15- to 35-nucleotide single-stranded molecules that can hybridize to specific corresponding sequences in a pre-mRNA.
• The clearest example of the potential of this strategy is provided by Duchenne muscular dystrophy (DMD).
• The goal of exon skipping in DMD is to convert a DMD mutation into an in-frame counterpart that generates a functional dystrophin, just as the deletions that allow the production of a partially functioning dystrophin are associated with the milder phenotype of Becker muscular dystrophy.

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The sequence specific binding of the exon-internal antisense oligonucleotide PRO051 interferes with the correct inclusion of exon 51 during splicing, so that the exon is actually skipped (B).

This restores the open reading frame of the transcript and allows the synthesis of a dystrophin similar to that in patients with Becker muscular dystrophy (BMD).

Schematic representation of exon skipping.

In a patient with Duchenne muscular dystrophy (DMD) who has a deletion of exon 50, an out-of-frame transcript is generated in which exon 49 is spliced to exon 51 (A). As a result, a stop codon is generated in exon 51, which prematurely aborts dystrophin synthesis.

An antisense oligonucleotides (ASO) with a central “gap” of DNA bases (gapmer ASO) binds to target mRNA by Watson‐Crick hybridization; RNase‐H1 recognizes an RNA–DNA heteroduplex, cleaving the target RNA strand selectively while leaving ASO strand intact to bind to additional target RNA.

An ASO modified to remove any potential to form RNA–DNA hybrids (non‐DNA‐like ASO) acts as a steric blocker to alter RNA maturation process, including modulation of splicing.

• Eteplirsen (or Exondys 51), an Antisense Oligonucleotide (AON) which triggers excision of exon 51 during pre-mRNA splicing of the dystrophin RNA transcript.
• Skipping exon 51 changes the downstream reading frame of dystrophin
• For DMD patients with particular frameshifting mutations, giving eteplirsen can restore the reading frame of the dystrophin mRNA and result in production of functional  dystrophin.

Eteplirsen: Approved for Duchenne Muscular Dystrophy

• It is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD.

Eteplirsen is beneficial for DMD patients with deletions ending at exon 50 and starting at exon 52. This covers ~20.5% of DMD patients with deletion mutations, or 14% of all DMD patients.

• Gene Editing

• The correction of a mutant gene sequence in its natural DNA context, in a sufficient number of target cells, would be an ideal treatment.
• This new technology, termed genome editing, uses engineered endonucleases containing a DNA-binding domain that will recognize a specific sequence in the genome, such as the sequence in which a missense mutation is embedded.
• Subsequently, a nuclease domain creates a double-stranded break, and cellular mechanisms for homology-directed repair (HDR) then repair the break, introducing the wild-type nucleotide to replace the mutant one.
• The template for the HDR must be based on a matching homologous wild-type DNA template that is introduced into the target cells before editing.
• The most widely used editing approach at present is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system, commonly referred to as CRISPR/Cas9.

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• In humans, genome editing offers possibilities for the correction of genetic defects in their natural genomic landscape, without the risks associated with the semirandom vector integration of some viral vectors used in gene therapy.

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• A major concern whose real dimensions are presently unknown is that the endonucleases can have off-target effects, which could cause mutations elsewhere in the genome.

NHEJ: Non-homologous end joining, HDR: Homology directed repair

Modification of the Somatic Genome by Transplantation

• Transplanted cells retain the genotype of the donor, and consequently transplantation can be regarded as a form of gene transfer therapy because it leads to a modification of the somatic genome.
• There are two general indications for the use of transplantation in the treatment of genetic disease.

• First, cells or organs may be transplanted to introduce wild-type copies of a gene into a patient with mutations in that gene. This is the case, for example, in homozygous familial hypercholesterolemia, for which liver transplantation is an effective but high-risk procedure.
• The second and more common indication is for cell replacement, to compensate for an organ damaged by genetic disease (for example, a liver that has become cirrhotic in α1-antitrypsin (deficiency).

• Stem Cell Transplantation

• Stem cells are defined by two properties:
1. their ability to proliferate to form the differentiated cell types of a tissue in vivo; and
2. their ability to self-renew—that is, to form another stem cell.

• Only three types of stem cells are in clinical use at present:
• Hematopoietic stem cells (HSCs), which can reconstitute the blood system after bone marrow transplantation;
• Corneal stem cells, which are used to regenerate the corneal epithelium, and
• Skin stem cells.
• These cells are derived from immunologically compatible donors.

Hematopoietic Stem Cell Transplantation in Non-storage Diseases

• In addition to its extensive application in the management of cancer, HSC transplantation using bone marrow stem cells is the treatment of choice for a selected group of monogenic immune deficiency disorders, including SCID of any type.
• Its role in the management of genetic disease in general, however, is less certain and under careful evaluation.
• For example, excellent outcomes have been obtained with allogeneic HSC transplantation in the treatment of children with β-thalassemia and sickle cell disease.

Hematopoietic Stem Cell Transplantation for Lysosomal Storage Diseases

• Bone marrow stem cell transplants are effective in correcting lysosomal storage in many tissues including, in some diseases, the brain, through the two mechanisms depicted in the figure.

Transplantation of Hematopoietic Stem Cells from Placental Cord Blood

• The discovery that placental cord blood is a rich source of HSCs is beginning to make a substantial impact on the treatment of genetic disease.
• The use of placental cord blood has three great advantages over bone marrow as a source of transplantable HSCs.

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• First, recipients are more tolerant of histoincompatible placental blood than of other allogeneic donor cells.Thus engraftment occurs even if as many as three HLA antigens, cell surface markers encoded by the major histocompatibility complex, are mismatched between the donor and the recipient.
• Second, wide availability of placental cord blood.
• Third, the risk for graft-versus-host disease is substantially reduced with use of placental cord blood cells.

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Liver Transplantation

• For some metabolic liver diseases, liver transplantation is the only treatment of known benefit.
• For example, the chronic liver disease associated with CF or α1AT deficiency can be treated only by liver transplantation, and together these two disorders account for a large fraction of all the liver transplants performed in the pediatric population.
• Liver transplantation has now been undertaken for more than two dozen genetic diseases. At present, the 5-year survival rate of all children who receive liver transplants is in the range of 70% to 85%.
• In the conditions in which hepatic damage has occurred (such as α1AT deficiency), the provision of healthy hepatic tissue restores growth and normal pubertal development.

Crigler Najjar type 1; Familial amyloid polyneuropathy; GSD, glycogen storage disease; hereditary haemochromatosis; haemolytic uraemic syndrome; organic acidurias; primary hyperoxalurias; progressive familial intrahepatic cholestasis; urea cycle disorders; Wilson’s disease.

The Problems and the Future of Transplantation

• First, the mortality after transplantation is still significant, and the morbidity from superimposed infection due to the requirement for immunosuppression and graft versus- host disease is substantial.
• Second, the finite supply of organs, cord blood being a singular exception.
• One solution to these difficulties involves the combination of stem cell and either genome editing or gene therapy.
• Here, a patient's own stem cells would be cultured in vitro and either transfected by gene therapy with the gene of interest or corrected by CRISPR/Cas9 editing and returned to the patient to repopulate the affected tissue with genetically restored cells.

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Induced Pluripotent Stem Cells

• The recently developed ability to induce the formation of pluripotent stem cells (iPSCs) from somatic cells has the potential to provide the optimal solution to both of the challenges of transplantation posed earlier.
• In this approach somatic cells, such as skin fibroblasts, would be taken from a patient in need of a transplant, and induced to form differentiated cells of the organ of interest.
• For example, the loss-of-function mutation in the α1-antitrypsin gene in the fibroblasts cultured from a patient with α1AT deficiency could be corrected, either by gene editing or gene therapy; the corrected cells could then be induced to form liver-specific iPSCs, which could then be transplanted into the liver of the patient to differentiate into hepatocytes.

• Alternatively, mature hepatocytes derived in vitro from the genetically corrected iPSCs could be transplanted.

• The great merit of this approach is that the genetically corrected liver cells are derived from the patient's own genome, thus evading immunological rejection of the transplanted cells as well as graft-versus-host disease.

• Substantial hurdles with iPSCs must first be overcome, however, including establishing the safety of transplanting cells derived by iPSC methodology and preventing epigenetic modifications in the derived cell type that are not characteristic of wild-type cells of the tissue of interest.

Gene Therapy

• Gene therapy is the introduction of a biologically active gene into a cell to achieve a therapeutic benefit.
• In 2012, the first gene therapy product was licensed in the United States and Europe for the treatment of lipoprotein lipase deficiency, and gene therapy has now been shown to be effective or extremely promising in clinical trials for almost a dozen inherited diseases, as in the next table.
• The goal of gene therapy is to transfer the therapeutic gene early enough in the life of the patient to prevent the pathogenetic events that damage cells.
• Moreover, correction of the reversible features of genetic diseases should also be possible for many conditions.

Examples of Inherited Diseases Treated by Gene Therapy of Somatic Tissues

PEG, polyethylene glycol; SCID, severe combined immunodefciency

In the preclinical studies, American Gene Technology (AGT) separated white blood cells from the HIV-positive patients’ blood using leukapheresis. They then expanded HIV-specific CD4 T-cells, then inserted a gene into those cells using the AGT103 lentivirus. The (miRNA) gene downregulates the CCR5 receptor, which disrupts the synthesis of proteins HIV needs to replicate. The modified cells are then infused back into the patient, where they remain in the body.

Essential Requirements of Gene Therapy for an�Inherited Disorder

1. Identity of the molecular defect
2. A functional copy of the gene: A complementary DNA (cDNA) clone of the gene or the gene itself must be available. If the gene or cDNA is too large for the current generation of vectors, a functional version of the gene from which nonessential components have been removed to reduce its size may suffice.
3. An appropriate vector : The most commonly used vectors at present are derived from the adeno-associated viruses (AAVs) or retroviruses, including lentivirus.
4. Knowledge of the pathophysiological mechanism of the disease.
5. Favorable risk-to-benefit ratio.
6. Appropriate regulatory components for the transferred gene. In thalassemia, for example, overexpression of the transferred gene would cause a new imbalance of globin chains in red blood cells, whereas low levels of expression would be ineffective.

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• In some enzymopathies, a few percent of normal expression may be therapeutic, and abnormally high levels of expression may have no adverse effect.

7. An appropriate target cell:

• Ideally, the target cell must have a long half-life or good replicative potential in vivo.
• It must also be accessible for direct introduction of the gene or, alternatively, it must be possible to deliver sufficient copies of the gene to it (e.g., through the bloodstream) to attain a therapeutic benefit.
• The feasibility of gene therapy is often enhanced if the target cell can be cultured in vitro to facilitate gene transfer into it; in this case, it must be possible to introduce a sufficient number of the recipient cells into the patient and have them functionally integrate into the relevant organ.

8. Strong evidence of efficacy and safety

9. Regulatory approval by an institutional review board or by a governmental agency

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General Considerations for Gene Therapy

• In the treatment of inherited disease, the most common use of gene therapy will be the introduction of functional copies of the relevant gene into the appropriate target cells of a patient with a loss-of-function mutation (because most genetic diseases result from such mutations).
• In these instances, precisely where the transferred gene inserts into the genome of a cell would, in principle, generally not be important.

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• If gene editing to treat inherited disease becomes possible, then correction of the defect in the mutant gene in its normal genomic context would be ideal and would alleviate concerns such as:
• The activation of a nearby oncogene by the regulatory activity of a viral vector, or
• The inactivation of a tumor suppressor due to insertional mutagenesis by the vector.

• In some long-lived types of cells, stable, long-term expression may not require integration of the introduced gene into the host genome.
• For example, if the transferred gene is stabilized in the form of an episome (a stable nuclear but non-chromosomal DNA molecule, such as that formed by an adeno-associated viral vector), and if the target cell is long-lived (e.g., T cells, neurons, myocytes, hepatocytes), then long-term expression can occur without integration.
• Gene therapy may also be undertaken to inactivate the product of a dominant mutant allele whose abnormal product causes the disease.
• For example, vectors carrying siRNAs could, in principle, be used to mediate the selective degradation of a mutant mRNA encoding a dominant negative proα1(I) collagen that causes osteogenesis imperfecta.

Gene Transfer Strategies

• An appropriately engineered gene may be transferred into target cells by one of two general strategies.
• The first involves introduction of the gene into cells that have been cultured from the patient ex vivo (that is, outside the body) and then reintroduction of the cells to the patient after the gene transfer.
• In the second approach, the gene is injected directly in vivo into the tissue or extracellular fluid of interest (from which it is taken up by the target cells).
• In some cases, it may be desirable to target the vector to a specific cell type; this is usually achieved by modifying the coat of a viral vector so that only the designated cells bind the viral particles.

The two major strategies used to transfer a gene to a patient

The Target Cell

• The ideal target cells are:
• stem cells (which are self-replicating) or
• Progenitor cells taken from the patient (thereby eliminating the risk for graft-versus-host disease).
• Both cell types have substantial replication potential.
• Introduction of the gene into stem cells can result in the expression of the transferred gene in a large population of daughter cells.
• At present, bone marrow is the only tissue whose stem cells have been successfully targeted as recipients of transferred genes. Genetically modified bone marrow stem cells have been used to cure two forms of SCID.
• Gene transfer therapy into blood stem cells is also likely to be effective for the treatment of hemoglobinopathies and storage diseases for which bone marrow transplantation has been effective.

• An important logistical consideration is the number of cells into which the gene must be introduced in order to have a significant therapeutic effect.
• To treat Phenylketonuria (PKU), for example, the approximate number of liver cells into which the phenylalanine hydroxylase gene would have to be transferred is approximately 5% of the hepatocyte mass, or approximately 10¹⁰ cells, although this number could be much less if the level of expression of the transferred gene is higher than wild type.
• A much greater challenge is gene therapy for muscular dystrophies, for which the gene must be inserted into a significant fraction of the huge number of myocytes in the body in order to have therapeutic efficacy.

DNA Transfer into Cells: Viral Vectors

• The ideal vector for gene therapy would be safe, readily made, and easily introduced into the appropriate target tissue, and it would express the gene of interest for life.
• Indeed, no single vector is likely to be satisfactory in all respects for all types of gene therapy, and a repertoire of vectors will probably be required.
• Here, we briefly review three of the most widely used classes of viral vectors, those derived from:
• retroviruses,
• adeno-associated viruses (AAVs), and
• adenoviruses.
• One of the most widely used classes of vectors is derived from retroviruses, simple RNA viruses that can integrate into the host genome. They contain only three structural genes, which can be removed and replaced with the gene to be transferred.
• The current generation of retroviral vectors has been engineered to render them incapable of replication.
• In addition, they are nontoxic to the cell, and only a low number of copies of the viral DNA (with the transferred gene) integrate into the host genome.

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• Moreover, the integrated DNA is stable and can accommodate up to 8 kb of added DNA.
• A major limitation of many retroviral vectors, however, is that the target cell must undergo division for integration of the virus into the host DNA, limiting the use of such vectors in nondividing cells such as neurons.
• In contrast, lentiviruses, the class of retroviruses that includes HIV, are capable of DNA integration in nondividing cells, including neurons.
• Lentiviruses have the additional advantage of not showing preferential integration into any specific gene locus, thus reducing the chances of activating an oncogene in a large number of cells.

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• Adeno-associated viruses (AAVs) do not elicit strong immunological responses, a great advantage that enhances the longevity of their expression.
• Moreover, they infect dividing or nondividing cells to remain in a predominantly episomal form that is stable and confers long-term expression of the transduced gene.
• A disadvantage is that the current AAV vectors can accommodate inserts of up to only 5 kb, which is smaller than many genes in their natural context.
• The third group of viral vectors, adenovirus-derived vectors, can be obtained at high titer, will infect a wide variety of dividing or nondividing cell types, and can accommodate inserts of 30 to 35 kb.
• However, in addition to other limitations, they have been associated with at least one death in a gene therapy trial through the elicitation of a strong immune response. At present their use is restricted to gene therapy for cancer.

Risks of Gene Therapy

• Gene therapy for the treatment of human disease has risks of three general types:

1. Adverse response to the vector or vector-disease combination.

2. Insertional mutagenesis causing malignancy.

The second concern is insertional mutagenesis, that is, that the transferred gene will integrate into the patient's DNA and activate a proto-oncogene or disrupt a tumor suppressor gene, leading possibly to cancer.

3. Insertional inactivation of an essential gene.

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Diseases That Have Been Amenable to Gene Therapy

• Although nearly a dozen single-gene diseases have been shown to improve with gene therapy, a large number of other monogenic disorders are potential candidates for this strategy, including retinal degenerations; hematopoietic conditions, such as sickle cell anemia and thalassemia; and disorders affecting liver proteins, such as PKU, urea cycle disorders, familial hypercholesterolemia, and α1AT deficiency.
• Gene therapy has been clearly effective for several disorders (X-linked SCID, metachromatic leukodystrophy, hemophilia B, and β-thalassemia) but also highlight some of the challenges associated with this therapeutic approach.

Severe X-Linked Combined Immunodeficiency

• The SCIDs are due to mutations in genes required for lymphocyte maturation.
• Affected individuals fail to thrive and die early in life of infection because they lack functional B and T lymphocytes.
• The most common form of the disease, X-linked SCID, results from mutations in the X-linked gene (IL2RG) encoding the γc-cytokine receptor subunit of several interleukin receptors.
• The receptor deficiency causes an early block in T- and natural killer–lymphocyte growth, survival, and differentiation and is associated with severe infections, failure to thrive, and death in infancy or early childhood if left untreated.
• The outcome of trials of X-linked SCID has been dramatic and resulted, in 2000, in the first gene therapy cure of a patient with a genetic disease.
• Bone marrow stem cells from the patients were infected in culture (ex vivo) with a retroviral vector that expressed the γc cytokine subunit cDNA. A selective advantage was conferred on the transduced cells by the gene transfer.

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• Transduced T cells and natural killer cells populated the blood of treated patients, and the T cells appeared to behave normally.
• Although the frequency of transduced B cells was low, adequate levels of serum immunoglobulin and antibody levels were obtained.
• Dramatic clinical improvement occurred, with resolution of protracted diarrhea and skin lesions and restoration of normal growth and development.
• This highly promising outcome, however, came at the cost of induction of a leukemia like disorder in 5 of the 20 treated patients, who developed an extreme lymphocytosis resembling T-cell acute lymphocytic leukemia; 4 of them are now well after treatment of the leukemia.
• The malignancy was due to insertional mutagenesis: the retroviral vector inserted into the LMO2 locus, causing aberrant expression of the LMO2 mRNA, which encodes a component of a transcription factor complex that mediates hematopoietic development.

• Current-generation vectors are designed to avoid this mutagenic effect by using strategies such as including a self-inactivating or “suicide” gene cassette in the vector to eliminate clones of malignant cells.
• At this point, bone marrow stem cell transplantation remains the treatment of choice for those children with SCID fortunate enough to have a donor with an HLA-identical match.
• For patients without such a match, autologous transplantation of hematopoietic stem and progenitor cells, in which the genetic defect has been corrected by gene therapy, offers a lifesaving alternative, but one that may not be without risk.

Precision Medicine: the Present and Future of the Treatment of Mendelian Disease

• The treatment of single-gene diseases embodies the concept of precision medicine tailored to the individual patient as deeply as any other area of medical treatment.
• Knowledge of the specific mutant sequence in an individual is central to many of the targeted therapies described in this chapter.
• The promise of gene therapy for an individual with a mendelian disorder must be based on the identification of the mutant gene in each affected individual and on the design of a vector that will deliver the therapeutic gene to the targeted tissue.
• Similarly, approaches based on gene editing require knowledge of the specific mutation to be corrected.
• Beyond this, however, precision medicine will frequently require knowledge of the precise mutant allele and of its specific effect on the mRNA and protein.

• In many cases, the exact nature of the mutation will define the drug that will bind to a specific regulatory sequence to enhance or reduce the expression of a gene.
• In other cases, the mutation will dictate the sequence of an allele-specific oligonucleotide to mediate the skipping of an exon with a premature termination codon, or of an siRNA to suppress a dominant negative allele.
• A compendium of small molecules will gradually become available to suppress particular stop codons, to act as chaperones that will rescue mutant proteins from misfolding and proteosomal degradation, or to potentiate the activity of mutant proteins.
• Genetic treatment is not only becoming more and more creative, it is becoming more and more precise.
• The future promises not only a longer life for many patients, but a life of vastly better quality.