Therapeutic Approaches for Treatment of Genetic Disorders: Tradition Leading to Evolution

Tanya Golani, RatnaDua Puri

Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, New Delhi

Correspondence to: Dr Ratna Dua Puri. Email: ratnadpuri@yahoo.com

 

 

Abstract

With more than 10,000 genetic disorders reported to date, the treatment offered to patients in the clinics is very limited. To develop a safe and efficacious treatment, it is important to understand the underlying pathophysiology and genetic etiology of the disease. This review aims to describe some available treatment approaches like conventional therapy, gene therapy, and stem cell therapy to treat these disorders. We also describe some of our clinical cases of different disease categories and their clinical management through these treatment approaches.

We structure this brief with an introduction followed by an overview of treatment modalities for genetic disorders available to date.

Keywords: Therapies, genetic disorders

Introduction

There are approximately 20,000 coding genes in the human genome that account for about 10,000 reported genetic disorders. These genetic diseases affect about 6-8% of the population worldwide. Although treatments have been in clinical trials for decades, very few, except inborn errors of metabolism, have successfully transitioned to the clinic (Barigga et al., 2021). To identify and develop potential therapies for patients, it is crucial to understand the pathophysiological cascade of the disorder and the molecular effect of the genetic mutation (Turnpenny et al., 2017). If the disease is recessive, providing the cell with a functional copy of the gene/protein will be enough to manage the disorder, however, if it is a dominant disorder with a gain of function mutation, blocking or disrupting the mutant gene will be necessary to correct the disorder at the gene level (Turnpenny et al., 2017). Despite vast challenges, the current era has witnessed a dramatic shift in successful treatments in the clinic, bringing hope to patients with genetic disorders. Within this context, we review the currently approved disease-modifying therapies and their mechanisms through specific patient case scenarios from the clinic.

Modalities of treatment for genetic disorders

Based on the etiopathophysiology, treatment approaches vary and can be divided into categories based on the level of intervention (Figure 1). These include symptomatic and supportive management; modifying the metabolic cassette by diet restrictions or special supplementation; providing deficient or mutant protein through protein, hormone, or enzyme replacement therapy; replacing abnormal protein - stem cell therapy, organ transplantation; correcting the genetic defect - gene therapy; and RNA based therapies (Gambello et al., 2018; Barigga et al., 2021).

1. Dietary manipulation

Inborn errors of metabolism (IEMs) are disorders caused by mutations affecting enzyme expression and function, leading to impaired metabolism (Gambello et al., 2018). There are around 116 treatable inborn metabolic disorders where timely treatment can prevent intellectual disability. For these disorders, the manipulation does not correct the basic defect but ameliorates the phenotype through pharmacological therapies (Pyeritz et al., 2021). The modalities of dietary modifications include:

• Restriction of the offending metabolite, e.g., phenylalanine in phenylketonuria, branched- chain amino acids in maple syrup urine disease (MSUD), galactose in galactosemia.
• Supplementation of deficient product, e.g., L- carnitine in systemic primary carnitine deficiency, L- arginine in urea cycle disorders (UCD).
• Vitamin/coenzyme supplementation, e.g., vitamin B12 in methylmalonic aciduria and homocystinuria (Cblc type), biotin in biotinidase deficiency.
• Stimulate an alternative metabolic pathway, e.g., carnitine in organic acidurias, glycine in isovaleric acidemia, penicillamine in Wilson disease, sodium benzoate and sodium phenylbutyrate in urea cycle disorders.
• Metabolic pathway inhibition, e.g., 2-(2-nitro-4-tri-fluorometylbenzoyl)-1,3-cyclohexanedione (NTBC) prevents the production of toxic metabolites by blocking the tyrosine metabolic pathway.

Table 1 depicts the pharmacological therapeutic options for patients with metabolic disorders of amino acids, lipids, and carbohydrates. Traditionally, though some metabolic disorders are treated using dietary modulation, many remain challenging to treat by these methods. For some IEMs, high-dose vitamins can help partially restore the enzymatic activity by raising cellular concentrations of cofactor as listed in Table 2. Other therapeutic options including enzyme replacement therapy (ERT), tissue transplantation, and gene therapy are discussed below.

Case study

A 1-month-old male child, second born to non-consanguineous parents, presented with complaints of lethargy, vomiting, and progressive encephalopathy. He was completely well till this presentation. On investigation, his ammonia levels were raised, and he had metabolic acidosis with an increased anion gap. Tandem mass spectrometry (TMS) showed increased propionylcarnitine (C3) and gas chromatography mass spectrometry (GCMS)of urine identified elevated propionylglycine, methylcitrate and 3-hydroxypropionate, suggestive of propionic acidemia. After initial emergency management and stabilization, he was treated with a protein-restricted diet and supplementation with isoleucine, valine, methionine, and threonine-free formula along with carnitine supplementation and metronidazole administration. He is on long-term follow-up and doing well.

2. Pathway modification:

Porphyrias are inherited disorders due to defects in the heme biosynthetic pathway. Treatment includes avoidance of triggers like exposure to sunlight and reduction in the amount of porphyrin in the body. Panhematin (hemin), an enzyme inhibitor, helps reduce porphyrin levels and is used for the treatment of acute intermittent porphyria (Pyeritz et al., 2021).

Vosoritide (trade name Voxzogo) is a recombinant C-type natriuretic peptide analog. It stimulates endochondral ossification by decreasing fibroblast growth factor receptor 3 (FGFR3) activity and thereby preventing inhibition of chondrocyte mineralization. It was approved by the United States Food and Drug Administration (FDA) in 2021 for use in patients with achondroplasia with open epiphyses, aged 5 years or above.

3. Replacement of Deficient Product:

This includes thyroid replacement in congenital hypothyroidism, packed red blood cells (RBCs) in thalassemia major, factor VIII in hemophilia A, factor IX in hemophilia B, and adrenocortical hormones in congenital adrenal hyperplasia (Turnpenny et al., 2017). Enzyme replacement therapy for inborn errors of metabolism is elaborated below.

3a. Enzyme Replacement Therapy (ERT)

Lysosomal storage disorders (LSDs) have been a paradigm for the treatment of genetic disorders with successful treatments spanning three decades. Replacement of the deficient lysosomal enzyme is the commonest therapeutic modality and currently, there are fourteen ERTs approved by regulatory authorities available for ten LSDs and many more are in clinical trials (Table 3) (Pogue et al., 2018). The enzyme requires efficient targeting to the lysosomes and over the years this has been achieved by enhancing the mannose -6 - phosphate receptors. The treatment is lifelong. Although ERT has been translational for the treatment of some disorders like Gaucher disease, there remain limitations to this treatment modality. The therapeutic enzyme may not reach specific regions like the central nervous system and skeletal system and have a limited impact on their disease process (Beck et al., 2018).

Beyond LSDs, ERT is used for adenosine deaminase (ADA)-deficient severe combined immunodeficiency. ERT with polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) is one of the modalities of treatment that allows the reduction of the accumulated substrate. The main indications for use are stabilization prior to hematopoietic stem cell transplantation (HSCT) or if there are contraindications to HSCT (Turnpenny et al., 2017). Additional disorders included recently in the armamentarium are Pegvaliase for phenylketonuria and trials are ongoing for other disorders (Table 3).

Case Study

A three-year child presents with failure to thrive, abdominal distention, and extreme irritability. He has been symptomatic since the last 6 months with multiple hospital visits. His parents are non-consanguineously married and there is no significant family history. His developmental milestones are normal except for a mild motor delay attributed to the massive abdominal distension. Examination identifies massive splenohepatomegaly with decreased growth parameters. Laboratory testing reveals anemia, thrombocytopenia, and decreased bone density with Erlenmeyer flask deformity on the femur radiograph. Eye examination is normal. Hematological disorders and infections are excluded, and he is diagnosed to have Gaucherdisease type 1 by glucocerebrosidase enzyme testing that shows a deficiency of the enzyme on a blood sample. The biallelic mutation is identified in the GBA gene - c.1603C>T (p.R496C) in exon 11. Definitive therapy with enzyme replacement therapy is initiated with the recombinant enzyme, imiglucerase. It is given as an intravenous infusion over two hours once every two weeks. Improvement in growth, decrease in the organomegaly, improved hematological parameters, and chitotriosidase biomarker is seen on follow-up. This child is now 14 years old with significantly improved disease parameters and activities and abilities like any other child of his age.

4. Drug treatment

This includes statins for familial hypercholesterolemia that reduce plasma low-density lipoprotein (LDL) through upregulation of the LDL receptors by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and endogenous cholesterol biosynthesis. Oral or intravenous bisphosphonates inhibit osteoclastic bone resorption and are used to increase bone mineral density in children and adults with osteogenesis imperfecta (Turnpenny et al., 2017). Everolimus (Afinitor) a small molecule drug is approved for the treatment of tuberous sclerosis complex (TSC)- associated partial-onset seizures in patients aged 2 years or above. It is an mTOR inhibitor that helps in tumor cell apoptosis and reduction in cell proliferation and angiogenesis, thereby inhibiting tumor cell growth in affected patients.

Proprotein convertase subtilisin/kexin type 9(PCSK9) inhibitors are a new class of drugs used for the reduction of low-density lipid-cholesterol levels in patients with hyperlipidemia (Turnpenny et al., 2017). They target and inactivate PCSK9 protease which attaches to LDL receptors leading to destruction in lysosomes. Evolocumab and Alirocumab are FDA-approved monoclonal antibodies for the treatment of patients with heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease that require LDL lowering. They are PCSK9 inhibitors that reduce LDL levels by 50-60% more than statins would do alone.

Hydroxyurea is an antineoplastic drug that is used for the treatment of patients with sickle cell anemia and thalassemia intermedia. It is known to reduce painful episodes and increase HbF levels. Through different mechanisms of action, it provides various therapeutic benefits to these patients. It is a potent ribonucleotide reductase (RR) inhibitor that inhibits DNA synthesis, by inhibiting the conversion of ribonucleotides to deoxyribonucleotides, resulting in cellular cytotoxicity, decrease in WBC, and elevated HbF levels, but this is still not fully understood.

5. Tissue transplantation:

This includes hematopoietic stem cell transplantation (HSCT) for many disorders including betathalassemia, inherited bone marrow failure syndromes, primary immune deficiency, e.g., severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, phagocytic cell defects (chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), severe congenital neutropenia), and familial hemophagocytic lymphohistiocytosis. Organ transplantation including liver, kidney, lung, and rarely heart transplantation is also performed. Liver transplantation can be done for the treatment of patients with urea cycle defects, tyrosinemia, Wilson disease, and MSUD (Turnpenny et al., 2017; Beck, 2018). A case synopsis of liver transplantation in our patient is described below.

Case Study

A 2.5 kg male neonate born at term presented with poor feeding and lethargy at day 5 of life. He developed seizures, dystonic posturing of limbs, encephalopathy, and respiratory distress. Urine ketones were positive with no metabolic acidosis. Investigations suggested the possibility of MSUD. The neonate was managed on a protein-restricted diet specific to MSUD and at the age of 22 months, India's first liver transplantation for MSUD was performed. The child is now 9 years of age and is doing well without any dietary restrictions.

6. Prenatal treatment:

Prenatal treatment of genetic disorders offers several benefits by treating inherited disorders early in life even before birth. Such early intervention reduces the period of irreversible damage to the affected organ (Turnpenny et al., 2017). An example is the treatment of a female fetus affected with congenital adrenal hyperplasia by prenatal administration of dexamethasone,in which virilization can be prevented by administration of dexamethasone, which suppresses fetal pituitary-adrenal axis, in small doses by the mother throughout pregnancy.

Lower urinary tract obstruction (LUTO) is a rare fetal condition that involves blockage in the urinary tract of the fetus which if left untreated can have severe complications and morbidity. Fetal interventions like vesicocentesis, vesico-amniotic shunting, and fetal cystoscopy are options of available treatment for LUTO.

Early fetal treatment interventions also include treatment of fetal arrhythmias like supraventricular tachyarrhythmia (SVT) and atrioventricular heart block. SVT is treated with antiarrhythmic medications like digoxin, flecainide, sotalol, and sometimes amiodarone. Depending on the etiology of the heart block involved, specific treatment including beta-sympathomimetics, immunoglobulin, apheresis, and/or fluorinated glucocorticoids can be incorporated. Percutaneous fetoscopy for the repair of open spina bifida has proven to be much more beneficial in comparison to postnatal treatment. Fetal therapy is reported for various other disorders like in utero stem cell transplantation in sickle cell disease, prenatal correction of X-linked hypohidrotic ectodermal dysplasia, and fetoscopic endotracheal occlusion (FETO) in fetuses with congenital diaphragmatic hernia.

7. Small Molecule Therapy

7a. Chaperone Therapy:

Genetic variations that impact protein structure and folding are degraded in the endoplasmic reticulum and proteosomes. Pharmacological chaperones are molecules that bind to the proteins and promote the correct folding of enzymes, thereby enhancing their function. Synthetic chaperones are used for the treatment of lysosomal storage disorders to correct target enzyme conformation. Though safe and efficacious, their use is limited due to their mutation specificity and undetermined optimal concentration on their inhibition activity (Beck, 2018). An example of chaperone therapy is Migalastat for amenable GLAvariants, in which the drug binds to the active site of enzyme alpha-galactosidase (the enzyme deficient in Fabry disease) leading to its stabilization and trafficking, increasing its catalytic activity (Beck, 2018). Small molecule modulators for cystic fibrosis include drugs that correct protein folding or enhance the cystic fibrosis transmembrane conductance regulator (CFTR) channel function. Lumacaftor, Tizacaftor and Elexacaftor are used independently or in combination for patients with the common F508del mutation. It assists to correct the protein configuration to enable appropriate cell trafficking of water and chloride. Ivacaftor is a potentiator that improves the transport of chloride ions through the ion channel by binding to the defective protein and increasing the open probability of the channel. They are approved for use in CF patients with specific mutations in the CFTR gene that control the gating of chloride ions across the plasma membrane (Turnpenny et al., 2017; Beck, 2018; Gambello et al., 2018).

7b. Substrate reduction therapy:

The aim of substrate reduction therapy (SRT) is to reduce the synthesis of the substrate or substrate precursor which are accumulated due to the enzyme deficiency. Advantages include oral administration, the potential to cross the blood-brain barrier and less immunogenicity (Beck, 2018). Examples of substrate inhibitors include N-butyldeoxynojirimycin (Zavesca®, Miglustat) for Gaucher type 1 and Eliglustat (Cerdelga). These drugs inhibit glucosylceramide synthase and limit the accumulation of the precursor, glucosylceramide. This enables the residual enzyme activity in the cell to metabolize the decreased substrate. Miglustat is approved for mild neuronopathic (type 3) Gaucher and non-neuronopathic (type 1) Gaucher disease. The gastrointestinal side effects limit the use of Miglustat for the approved indications. The second sanctioned oral therapy for Gaucher disease, Eliglustat (Cerdelga), is approved for use in adult Gaucher disease type 1 [with known CYP2D6 genotype]. Genistein, a naturally occurring isoflavone, inhibits the synthesis of glycosaminoglycans and was proposed for the treatment of mucopolysaccharidosis (MPS) type III. However long-term data on efficacy is lacking for this drug (Beck, 2018; Gambello et al., 2018).

7c. Stop-codon read-through enhancers:

Small molecules which modulate splicing and enhance stop-codon read-through have become a popular approach for the treatment of genetic disorders like spinal muscular atrophy (SMA) (Beck et al., 2018). Risdiplam (brand name Evrysdi) is a small molecule SMN2 enhancer which is taken orally daily by SMA patients. This splicing modifier increases the SMN protein expression by preventing exon 7 of the SMN2 gene from getting spliced thereby causing a decrease in exon 7 deleted SMN protein and increasing the production of full-length SMN protein. Ataluren (brand name Translarna) enables read-through of the premature stop codon to produce full-length dystrophin protein or protein larger than the mutated version. It results in a Becker-type phenotype and is used for Duchenne muscular dystrophy (DMD) patients aged 5 years or above.

Case Study

A 15-month-old male child, first born to non-consanguineous parents, came to medical attention with complaints of being unable to hold neck or roll over, limited limb movements, and poor speech. On examination he had hypotonia, areflexia and tongue fasciculations. He was diagnosed with SMA type 1 and was started on Risdiplam (2.7 ml per day) and after 5 months he showed improvements in limb movements, swallowing and speech.

8. RNA modifications:

These therapies are based on mRNA modification with suppression of mRNA levels or correcting the function of mRNA and include antisense oligonucleotide-based therapy and RNA interference.

8a. Antisense Oligonucleotide (ASO):

ASOis a therapeutic strategy in which a short sequence-specific single-stranded antisense oligonucleotide (usually 8-30 bases in length) binds to the target mRNA inhibiting its gene expression at the protein level (Figure 2) (Turnpenny et al., 2017). Table 4 shows some ASO-based treatments that are approved for use by the US FDA. Although advances made in developing ASO have considerably impacted patient management, a key challenge is that each ASO is mutation specific. Therefore, developing a therapy for a rare mutation can be expensive (Shahryariet al., 2019).

Case Study

A 5-year-old male child, born to non-consanguineous parents, was evaluated in our clinic with delayed motor milestones and subsequently was diagnosed with SMA type 2 at the age of 15 months. There was history of an episode of respiratory infection which was managed at home. The child was selected to receive intrathecal Nusinersen through the humanitarian access program and has received 4 doses to date. He is currently showing improved neck and hand control as well as overall well-being.

8b. RNA interference (RNAi)

This therapy, unlike ASO (being bound to target mRNA) targets the gene and cleaves off the mRNA with resultant gene silencing. It is 1000-fold more efficacious than ASO therapy. The synthetic double-stranded RNA sequences called small interfering RNAs (siRNAs) bind to targeted mRNA resulting in their RISC-associated cleavage. This therapy is of special interest when gene knockdown at a specific target is desired (Figure 3). Examples of RNAi therapy are Patisiran for hereditary amyloidosis and Lumasiran for primary hyperoxaluria type 1 (PH1) (Table 4) (Turnpenny et al., 2017; Shahryari et al., 2019).

9. Stem Cell Therapy:

Stem cells are unspecialized cells that have the capacity for self-renewal and the ability to differentiate into specialized cells of many lineages upon proper stimulation. Stem cells can be embryonic stem cells, which are pluripotent cells derived from the inner cell mass of the blastocyst and can differentiate into derivatives of all three germ layers i.e., ectoderm, endoderm, and mesoderm. Somatic stem cells, now called induced pluripotent stem cells, are cells capable of self-renewal and can differentiate into cell types of tissues from which they are derived (Turnpenny et al., 2017; Maldonado et al., 2021).

9a. Hematopoietic stem cell transplantation

HSCT is a promising treatment approach owing to the accessibility of the hematopoietic cells, their well-described behavior and ability to survive ex-vivo manipulation. The rationale of transplantation is the introduction of healthy cells that would be internalized in the donor to allow enzyme production (Maldonado et al., 2021). The recipient hematopoietic system is repopulated with the healthy donor cells that can produce the deficient enzyme lifelong. Efficacy is proved for mucopolysaccharidosis (MPS) type I, metachromatic leukodystrophy (MLD), and X-linked adrenoleukodystrophy (ALD). Premedication with ERT prior to transplant in patients of MPS I is reported to improve transplant outcomes. The current hematopoietic stem cell transplantation (HSCT) strategies involve a healthy donor and allogenic transplant with its attending limitations. Genetic disorders where HSCT is an approved therapeutic modality include primary immune deficiencies, hemoglobinopathies, bone marrow failure syndromes, lysosomal disorders including MPS-1, alpha mannosidosis,presymptomatic, late-onset and slowly progressing MLD, and presymptomatic cerebral X-linked ALD (Beck, 2018; Gambello et al., 2018; Maldonado et al., 2021).

10. Gene therapy:

Gene therapy is a therapeutic approach to correct defective gene function by replacing or targeted editing of the defective gene. Gene editing through zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9 are the most used methods (Gupta et al., 2015). Sickle cell disease and Leber congenital amaurosis are some recent examples of use of gene editing for definitive treatment (Shahryari et al., 2019).

Gene therapy trials in humans began in 1990, but wide inroads have only been made in the last decade when the first human gene therapy, Luxturna for inherited retinal atrophy was approved by the US FDA. Despite immense therapeutic implications for the treatment of genetic disorders, there remain challenges that limit easy transition to the clinic (Turnpenny et al., 2017). These include safety and efficacy, product interaction with the host cells, need for prolonged clinical laboratory studies, high cost of therapeutic products, and immune response to transgene or associated vector (Shahryari et al., 2019). The two mechanisms for transgene transfer include ex vivo and in vivo therapy (Figure 4). Based on the genetic disorders to be treated, gene therapies are limited to different target organs involving specific gene transfer methods and suitable vectors. Type of gene therapy are subdivided into viral-mediated and non-viral mediated gene therapy (Turnpenny et al., 2017).

Gene therapy in autologous patient-derived progenitor cells and ex vivo gene therapy are alternative approaches that limit graft vs host disease (GVHD). The most recent gene therapy to be approved is Zynteglo [Betibeglogeneautotemcel (beti-cel)], a lentiviral associated βA(T87Q)-globin gene sequence for use in children 12 years or older with transfusion-dependent beta-thalassemia. This is an autologous transplant with ex vivo gene therapy via a lentiviral vector with genetic modification of the autologous hematopoietic stem cells (Beck, 2018; Shahryari et al., 2019). Recently, RoctavianTM (Valoctocogene roxaparvovec) has been granted conditional marketing approval by the European Commission (EC) for treatment of severe hemophilia A in adult patients without a history of Factor VIII inhibitors and absence of detectable antibodies to adeno-associated virus sero type 5 (AAV5). This is the first approved single-time infusion therapy for hemophilia A. It delivers the functional copy of the gene enabling the body to produce Factor VIII.

10a. Viral vectors:

These are commonly used for gene therapy currently. Their benefits include high efficiency of transduction of transgene to the host, specific and targeted gene therapy, safety and efficacy, and reduced administration of doses. The common viral vectors used include retrovirus, lentivirus, adenovirus, and adeno-associated virus (Table 5) (Turnpenny et al., 2017).

10b. Non-viral vector therapy:

These are liposome-mediated DNA transfers. This is safer and unlikely to elicit an immune response. However, they have a low efficacy with transient expression of a transgene. However, they can incorporate a large sequence of DNA for delivery to the target cell. Nanoparticles are alternative synthetic vectors that offer similar benefits to liposome-mediated gene transfer. The efficacy and expression studies are still under investigation and a topic of debate (O'Connor et al., 2006; Turnpenny et al., 2017).

Case study

A 15-month-old male born to non-consanguineous parents presented with weakness in both lower limbs since infancy and an inability to stand independently. His social and language milestones were age appropriate. He was diagnosed with SMA type 1 having a homozygous deletion of exon 7 in the SMN1 gene. The child was enrolled in the AVXS-101 GMAP gene therapy study and meanwhile was given regular physiotherapy. He successfully received Zolgensma as an intravenous single-time dose. Post 8 months he is now able to feed himself and stand with support for some time.

Gene therapy possesses a relative advantage of attaining a tag of an orphan drug. However, it is important that the transgene must survive and proliferate in the host without eliciting any negative immune responses (Shahryari et al., 2019). Table 6 shows few approved gene therapies for treatment of genetic disorders.

Conclusion

With advancements in medical science and the approval of various therapies, the treatment of various genetic diseases is now possible through various approaches (Table 7). With different gene editing and transfer methods available, we can practically rewrite the genetic code inside the cell with innumerable options. However, to develop a successful curative treatment, it is vital to further understand the cellular and genetic pathology of each disorder (Turnpenny et al., 2017). Additionally, it is important to address other technical and ethical issues while developing a genetic treatment to increase its safety and efficacy. The ratio of benefits to risks must always be acceptable and beneficial for patients. Previously untreatable disorders like Duchenne muscular dystrophy and spinal muscular atrophy now have efficacious therapies, indicating that in the coming days more therapies will follow. Despite the approval of many therapies for rare disorders, the impact on short and long-term outcomes requires follow-up and definite criteria to demonstrate a detailed impact on the natural history of the disorder.

References

1. Barigga A, et al. Management of genetic diseases: present and future. Rev FacMed Hum. 2021;21(2):399-416.

2. Beck M. Treatment strategies for lysosomal storage disorders. Dev Med Child Neurol. 2018;60(1):13-18.

3. Gambello MJ, et al. Current strategies for the treatment of inborn errors of metabolism.J Genet Genomics. 2018; 45(2):61-70.

4. Gupta A, et al. Genome Editing: Precise and "CRISPER". Genetic Clinics. 2015;8(3):10-14.

5. Maldonado R, et al. Curative gene therapies for rare diseases. J Community Genet. 2021;12(2):267-276.

6. O'Connor TP, et al. Genetic medicines: treatment strategies for hereditary disorders. Nat Rev Genet. 2006:7(4):261-276.

7. Pogue RE, et al. Rare genetic diseases: update on diagnosis, treatment, and online resources. Drug Discov. 2018;23(1):187-195.

8. Pyeritz R, et al. In: Emery and Rimoin's Principles and Practice of Medical Genetics and Genomics: Metabolic disorders. Editors: Pyeritz R, Korf B,Grody W. Seventh edition. 2021. Elsevier.

9. Shahryari A, et al. Development and clinical translation of approved gene therapy products for genetic disorders.Front Genet. 2019;10:868.

10. Turnpenny P, et al. In: Emery's Elements of Medical Genetics. Fifteenth edition. 2017. p 200-214. Elsevier.