Mutation-Directed Therapeutics for Neurofibromatosis Type I
2020; Cell Press; Volume: 20; Linguagem: Inglês
10.1016/j.omtn.2020.04.012
ISSN2162-2531
AutoresAndré Leier, David M. Bedwell, Ann T. Chen, George Dickson, Kim M. Keeling, Robert A. Kesterson, Bruce R. Korf, Tatiana T. Marquez Lago, Ulrich F. Müller, Linda Popplewell, Jiangbing Zhou, Deeann Wallis,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoSignificant advances in biotechnology have led to the development of a number of different mutation-directed therapies. Some of these techniques have matured to a level that has allowed testing in clinical trials, but few have made it to approval by drug-regulatory bodies for the treatment of specific diseases. While there are still various hurdles to be overcome, recent success stories have proven the potential power of mutation-directed therapies and have fueled the hope of finding therapeutics for other genetic disorders. In this review, we summarize the state-of-the-art of various therapeutic approaches and assess their applicability to the genetic disorder neurofibromatosis type I (NF1). NF1 is caused by the loss of function of neurofibromin, a tumor suppressor and downregulator of the Ras signaling pathway. The condition is characterized by a variety of phenotypes and includes symptoms such as skin spots, nervous system tumors, skeletal dysplasia, and others. Hence, depending on the patient, therapeutics may need to target different tissues and cell types. While we also discuss the delivery of therapeutics, in particular via viral vectors and nanoparticles, our main focus is on therapeutic techniques that reconstitute functional neurofibromin, most notably cDNA replacement, CRISPR-based DNA repair, RNA repair, antisense oligonucleotide therapeutics including exon skipping, and nonsense suppression. Significant advances in biotechnology have led to the development of a number of different mutation-directed therapies. Some of these techniques have matured to a level that has allowed testing in clinical trials, but few have made it to approval by drug-regulatory bodies for the treatment of specific diseases. While there are still various hurdles to be overcome, recent success stories have proven the potential power of mutation-directed therapies and have fueled the hope of finding therapeutics for other genetic disorders. In this review, we summarize the state-of-the-art of various therapeutic approaches and assess their applicability to the genetic disorder neurofibromatosis type I (NF1). NF1 is caused by the loss of function of neurofibromin, a tumor suppressor and downregulator of the Ras signaling pathway. The condition is characterized by a variety of phenotypes and includes symptoms such as skin spots, nervous system tumors, skeletal dysplasia, and others. Hence, depending on the patient, therapeutics may need to target different tissues and cell types. While we also discuss the delivery of therapeutics, in particular via viral vectors and nanoparticles, our main focus is on therapeutic techniques that reconstitute functional neurofibromin, most notably cDNA replacement, CRISPR-based DNA repair, RNA repair, antisense oligonucleotide therapeutics including exon skipping, and nonsense suppression. Neurofibromatosis type I (NF1, OMIM #162200) is one of the most common genetic disorders, occurring in approximately 1:2,000–3,000 births.1Friedman J.M. Gutmann D.H. MacCollin M. Riccardi V.M. Neurofibromatosis: Phenotype, Natural History, and Pathogenesis. Third Edition. Johns Hopkins University Press, 1999Google Scholar,2Kallionpää R.A. Uusitalo E. Leppävirta J. Pöyhönen M. Peltonen S. Peltonen J. Prevalence of neurofibromatosis type 1 in the Finnish population.Genet. Med. 2018; 20: 1082-1086Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar It is caused by pathogenic variants in the NF1 gene (NCBI: NG_009018.1), which is located on chromosome 17q11.2. With a length of about 300 kb, it is one of the largest human genes. Precursor mRNA (pre-mRNA) splicing of its 61 exons is complex, with several alternatively spliced exons, alternative splice sites, and potentially regulatory pseudo-exon inclusion.3Buratti E. Baralle D. Exon skipping mutations in neurofibromatosis.Methods Mol. Biol. 2012; 867: 65-76Crossref PubMed Scopus (4) Google Scholar, 4Vandenbroucke I. Callens T. De Paepe A. Messiaen L. Complex splicing pattern generates great diversity in human NF1 transcripts.BMC Genomics. 2002; 3: 13Crossref PubMed Scopus (0) Google Scholar, 5Thomson S.A. Wallace M.R. RT-PCR splicing analysis of the NF1 open reading frame.Hum. Genet. 2002; 110: 495-502Crossref PubMed Scopus (26) Google Scholar The NF1 mRNA transcript variant 2 (NCBI: NM_000267.3) has a length of approximately 12.4 kb and is composed of 57 constitutively expressed exons. Neurofibromin (P21359-2), the protein encoded by NF1, is a tumor suppressor and downregulator of the Ras signaling pathway. It is expressed in many cell types, notably in neurons, Schwann cells, oligodendrocytes, astrocytes, and leukocytes. Loss of function, or lack of neurofibromin, leads to a condition that is characterized by a pleiotropic phenotype affecting the skin (café-au-lait macules, skin fold freckling, hyperpigmentation, cutaneous neurofibromas), the eye (Lisch nodules and optic glioma), skeleton (dysplasias and scoliosis), and peripheral and central nervous system (CNS) (cognitive disabilities, motor delays, gliomas, neurofibromas). Malignant peripheral nerve sheath tumors (MPNSTs) can be observed, with poor prognosis. Almost 2,900 pathogenic variants have been reported in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). To date, NF1 cannot be cured. Treatments attempt to manage the various symptoms, especially NF1-associated tumors and other cancers, using standard cancer therapies. At least a dozen different therapeutics targeting almost as many proteins have been used in clinical trials for NF-associated tumors.6Lin A.L. Gutmann D.H. Advances in the treatment of neurofibromatosis-associated tumours.Nat. Rev. Clin. Oncol. 2013; 10: 616-624Crossref PubMed Scopus (58) Google Scholar,7Gutmann D.H. Blakeley J.O. Korf B.R. Packer R.J. Optimizing biologically targeted clinical trials for neurofibromatosis.Expert Opin. Investig. Drugs. 2013; 22: 443-462Crossref PubMed Scopus (63) Google Scholar Also, oncolytic viral therapies for treating MPNSTs have shown promising results.8Antoszczyk S. Rabkin S.D. Prospect and progress of oncolytic viruses for treating peripheral nerve sheath tumors.Expert Opin. Orphan Drugs. 2016; 4: 129-138Crossref PubMed Scopus (2) Google Scholar However, these treatments are largely adaptations of existing therapies against sporadic tumors and do not target the specific growth control pathways dysregulated in NF1. More recently, drug development has focused on targeting key molecules downstream of NF1, most notably the RAF-MEK-ERK signaling cascade downstream of RAS.9Baker N.M. Der C.J. Cancer: drug for an "undruggable" protein.Nature. 2013; 497: 577-578Crossref PubMed Scopus (39) Google Scholar, 10Kim A. Dombi E. Tepas K. Fox E. Martin S. Wolters P. Balis F.M. Jayaprakash N. Turkbey B. Muradyan N. et al.Phase I trial and pharmacokinetic study of sorafenib in children with neurofibromatosis type I and plexiform neurofibromas.Pediatr. Blood Cancer. 2013; 60: 396-401Crossref PubMed Scopus (57) Google Scholar, 11Dombi E. Baldwin A. Marcus L.J. Fisher M.J. Weiss B. Kim A. Whitcomb P. Martin S. Aschbacher-Smith L.E. Rizvi T.A. et al.Activity of selumetinib in neurofibromatosis type 1-related plexiform neurofibromas.N. Engl. J. Med. 2016; 375: 2550-2560Crossref PubMed Scopus (363) Google Scholar, 12Jessen W.J. Miller S.J. Jousma E. Wu J. Rizvi T.A. Brundage M.E. Eaves D. Widemann B. Kim M.O. Dombi E. et al.MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors.J. Clin. Invest. 2013; 123: 340-347Crossref PubMed Scopus (223) Google Scholar, 13Watson A.L. Anderson L.K. Greeley A.D. Keng V.W. Rahrmann E.P. Halfond A.L. Powell N.M. Collins M.H. Rizvi T. Moertel C.L. et al.Co-targeting the MAPK and PI3K/AKT/mTOR pathways in two genetically engineered mouse models of Schwann cell tumors reduces tumor grade and multiplicity.Oncotarget. 2014; 5: 1502-1514Crossref PubMed Google Scholar, 14Jousma E. Rizvi T.A. Wu J. Janhofer D. Dombi E. Dunn R.S. Kim M.O. Masters A.R. Jones D.R. Cripe T.P. Ratner N. Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of Neurofibromatosis type 1.Pediatr. Blood Cancer. 2015; 62: 1709-1716Crossref PubMed Scopus (21) Google Scholar MEK inhibitors such as selumetinib have demonstrated effectiveness for patients who respond and can tolerate treatment; however, not all patients benefit, plexiform neurofibromas do not completely disappear, and there can be significant side effects.11Dombi E. Baldwin A. Marcus L.J. Fisher M.J. Weiss B. Kim A. Whitcomb P. Martin S. Aschbacher-Smith L.E. Rizvi T.A. et al.Activity of selumetinib in neurofibromatosis type 1-related plexiform neurofibromas.N. Engl. J. Med. 2016; 375: 2550-2560Crossref PubMed Scopus (363) Google Scholar Hence, therapeutics that address the underlying cause of the disease by restoring neurofibromin function to a level that leads to a non-pathogenic phenotype do not yet exist. Various gene and mRNA targeting strategies have been proposed in the context of other diseases. For example, Zhou et al.15Zhou L.Y. Qin Z. Zhu Y.H. He Z.Y. Xu T. Current RNA-based therapeutics in clinical trials.Curr. Gene Ther. 2019; 19: 172-196Crossref PubMed Scopus (57) Google Scholar specifically reviewed many of the RNA-based therapeutics in clinical trials. These and other strategies are worth being evaluated for their therapeutic potential in NF1. In the following sections we introduce therapeutic strategies to replace or repair the NF1 gene or its transcripts or to inhibit the effect of certain genetic mutations. Table 1 provides a summary of these methods, the types of mutations that can be targeted, advantages and disadvantages, as well as examples of success for each method. We discuss gene replacement by cDNA/mRNA delivery, CRISPR (clustered regularly interspaced short palindromic repeats)-based DNA repair, RNA repair, exon skipping, and nonsense suppression therapies (in combination with inhibition of nonsense-mediated mRNA decay [NMD]) in the context of NF1 and address the pros and cons of these individual approaches. Subsequently, we evaluate data regarding required levels of neurofibromin function. Lastly, we address important aspects of the delivery of any such therapeutic and nanoparticles as a promising delivery vehicle. We anticipate that this review of mutation-directed therapeutics for NF1 may also stimulate similar discussions and active steps toward the development of treatments of other genetic diseases.Table 1Approaches for NF1 Gene TherapyApproachTargeted MutationsAdvantagesDisadvantages/ChallengesSuccessesNF1 StatusGene replacementall loss of function mutationsmight target the largest mutation spectrummNF1 cDNA; efficient delivery using nanoparticlesLuxterna for retinal dystrophy associated with loss of RPE65, and Zolgensma for SMA and loss of SMN1development of full-length mNf1 cDNA and development of model systems for testingGenome editingmost small mutationspermanent cell editingefficiency of editing; non-specific gene editing; delivery using nanoparticlesex vivo CCR5 deletion to block HIV infectiontesting CRISPR-Cas9 and CRISPR Prime in nanoparticles and development of model systems for testingRNA editingmost small mutations in 5′ and 3′ regionsdoes not change DNAefficiency of editing, non-permanence; delivery using virus or nanoparticlesβ-globin and DMPK repair in vitrodefining high-efficiency splice sites within NF1 and evolving ribozymes and development of model systems for testingExon skippingselect exonslow toxicityeach exon must be considered/designed separately; delivery using cell-penetrating peptidesExondys 51 (eteplirsen) and Vyondys 53 for DMD; Spinraza for SMAdefinition of selected exons and testing of AOs and development of model systems for testingNSTnonsense ~20%low toxicity; may be able to repurpose other drugsefficiency of readthrough; NMDAtaluran for cystic fibrosissmall molecule drug screens for NSTs and development of model systems for testing Open table in a new tab Perhaps one of the more straightforward concepts of gene therapy is to supplement a working copy of a gene into a cell with a defective gene. This allows for the treatment of gene deletions or other loss-of-function type mutations. In 2017, Luxturna (voretigene neparvovec) became the first US Food and Drug Administration (FDA)-approved gene replacement therapy for the treatment of patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy that leads to vision loss and may cause complete blindness in certain patients. Luxturna works by delivering a normal copy of the RPE65 gene directly to retinal cells, using a recombinant adeno-associated virus serotype 2 (rAAV2) vector. This therapy is regarded as the first true FDA-approved gene therapy, because the viral treatment is delivered directly to the patient's body. That said, Luxturna, even though heralded as a therapy that restores vision, is far from being a cure: it has shown to improve vision to some degree in some but not all treated patients. Furthermore, improvements may not be permanent.16Darrow J.J. Luxturna: FDA documents reveal the value of a costly gene therapy.Drug Discov. Today. 2019; 24: 949-954Crossref PubMed Scopus (88) Google Scholar Previous FDA-approved gene therapies (Yescarta [axicabtagene ciloleucel] and Kymriah [tisagenlecleucel]) are called ex vivo treatments, as the cells receiving gene therapy (immune cells) are removed from the body prior to treatment. Zolgensma (onasemnogene abeparvovec) is another therapeutic that uses an AAV to deliver a functional copy of the SMN1 gene to motor neurons in pediatric patients with spinal muscular atrophy (SMA). Gene replacement for NF1 has been challenging for two primary reasons: the lack of a full-length NF1 cDNA and a delivery system. Since discovery of the NF1 gene in 1990, research efforts have been hindered by the lack of a full-length coding cDNA. This is due to the size of the cDNA and toxicity of the human construct. Despite this, as the GAP-related domain (GRD) was assumed to be the most important segment of NF1, replacement with the GRD has been attempted, but was unsuccessful in several ways. Expression of isolated GRD is unable to rescue overgrowth of neural crest-derived tissues, leading to perinatal lethality in Nf1−/− embryos.17Ismat F.A. Xu J. Lu M.M. Epstein J.A. The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1 mice.J. Clin. Invest. 2006; 116: 2378-2384PubMed Google Scholar The GRD rescues endothelial but not neural crest development in Nf1 mice. These results suggest that neurofibromin may possess activities outside of the GRD that modulate neural crest homeostasis and that therapeutic approaches solely aimed at targeting Ras activity may not be sufficient. Most recently, the feasibility of restoring Ras guanosine triphosphatase (GTPase) via exogenous expression of various NF1-GRD constructs, via gene delivery using a panel of AAV vectors in MPNST and human Schwann cells (HSCs), has been explored.18Bai R.Y. Esposito D. Tam A.J. McCormick F. Riggins G.J. Wade Clapp D. Staedtke V. Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors.Gene Ther. 2019; 26: 277-286Crossref PubMed Scopus (19) Google Scholar Several AAV serotypes achieved favorable transduction efficacies, in particular AAV-DJ. A membrane-targeting GRD fused with an H-Ras C-terminal motif containing the palmitoylation sites and CAAX motif (C10) inhibited the Ras pathway and MPNST cells in a NF1-specific manner; however; a transfection efficiency of only 9.8% of cells at a MOI of 5,000 is reported. Since the non-transduced populations exhibit a clear growth advantage, efficacy would require almost all of the MPNST cells or the majority of the NF1-haploid Schwann cells to receive the GRD-C10 transgene. With the current available tools for in vivo gene delivery, this would only be feasible with a drastically improved AAV vector for MPNST or Schwann cells, which could be achieved through protein engineering of the AAV capsids. We have developed and validated a mouse Nf1 cDNA expression system that allows us to examine the biochemical effects of any Nf1 genetic variant.19Wallis D. Li K. Lui H. Hu K. Chen M.J. Li J. Kang J. Das S. Korf B.R. Kesterson R.A. Neurofibromin (NF1) genetic variant structure-function analyses using a full-length mouse cDNA.Hum. Mutat. 2018; 39: 816-821Crossref PubMed Scopus (16) Google Scholar The full-length cDNA sequences of endogenous hNF1 and mNf1 have 92% sequence identity; amino acid sequences share 98% identity.20Anastasaki C. Le L.Q. Kesterson R.A. Gutmann D.H. Updated nomenclature for human and mouse neurofibromatosis type 1 genes.Neurol. Genet. 2017; 3: e169Crossref PubMed Scopus (15) Google Scholar A heavily codon-optimized human NF1 cDNA21Sherekar M. Han S.W. Ghirlando R. Messing S. Drew M. Rabara D. Waybright T. Juneja P. O'Neill H. Stanley C.B. et al.Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.J. Biol. Chem. 2020; 295: 1105-1119Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar (R777-E139 Hs.NF1 [Addgene plasmid #70423]) that is stable in E. coli and non-toxic to human cells has also become available. This human construct is so heavily codon optimized that when the sequence is used for a BLASTn (nucleotide) search, the NF1 gene sequence does not come back as a hit; yet a BLASTp (protein) search comes back with 100% identity. Furthermore, GC content is increased from 43% to 60% in the codon-optimized transcript. Hence, the mNf1 cDNA is more homologous to the endogenous human sequence. Furthermore, codon optimization may have direct implications, as codon usage is thought to affect transcription and translation efficiency as well as mRNA stability and protein folding. Another hNF1 cDNA eliminates the cDNA cloning toxicity by introducing a mini-intron.22Cui Y. Morrison H. Construction of cloning-friendly minigenes for mammalian expression of full-length human NF1 isoforms.Hum. Mutat. 2019; 40: 187-192Crossref PubMed Scopus (2) Google Scholar Gene Replacement for NF1. As viral vectors cannot accommodate the size or the NF1 cDNA, we are investigating the ability to package our full-length mouse Nf1 cDNA into nanoparticles for intracellular delivery as described in "Delivery of NF1 Gene Therapeutic" below. As an NF1 therapeutic, this approach would target the largest mutation spectrum. Direct repair of small DNA or RNA mutations is an alternative to whole-gene replacement. The three prominent systems for DNA repair based on programmable nucleases are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR and CRISPR-associated (Cas) proteins. The CRISPR-Cas9 system has demonstrated great promise in many biological applications.23Heidenreich M. Zhang F. Applications of CRISPR-Cas systems in neuroscience.Nat. Rev. Neurosci. 2016; 17: 36-44Crossref PubMed Google Scholar It is an RNA-based programmable nuclease derived from a prokaryotic immune system. The CRISPR-Cas9 nuclease system has emerged as the most promising genome editing technology. The system uses an engineered single guide RNA (sgRNA) to direct the Cas9 nuclease to complementary regions, where Cas9 cleaves the recognized DNA and generates double-stranded breaks (DSBs), leading to insertions or deletions at specific target genomic loci (Figure 1).24Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. 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Dermatol. 2016; 136: e87-e93Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar When an externally supplied homologous donor template is provided, HDR may enable precise genome editing and permanent correction of a targeted mutation.27Morgan M.A. Lawrence T.S. Molecular pathways: overcoming radiation resistance by targeting DNA damage response pathways.Clin. Cancer Res. 2015; 21: 2898-2904Crossref PubMed Scopus (153) Google Scholar However, under normal physiological conditions, NHEJ repair, which is an error-prone process and tends to lead to disruption of targeted genes is more than 10-fold more frequent than HDR at DSBs.27Morgan M.A. Lawrence T.S. Molecular pathways: overcoming radiation resistance by targeting DNA damage response pathways.Clin. Cancer Res. 2015; 21: 2898-2904Crossref PubMed Scopus (153) Google Scholar Consequently, without suppressing NHEJ, the HDR-mediated precise genome editing often suffers from low efficiency. 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Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (1476) Google Scholar directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. Prime editing offers efficiency and product purity advantages over HDR with much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. One of the earliest studies utilizing CRISPR-Cas9 for somatic in vivo repair editing was performed in a mouse model for tyrosinemia to correct a mutation in the enzyme fumarylacetoacetate hydrolase in hepatocytes.30Yin H. Xue W. Chen S. Bogorad R.L. Benedetti E. Grompe M. Koteliansky V. Sharp P.A. Jacks T. Anderson D.G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.Nat. 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Another trial proposes to treat hematopoietic stem cells of sickle cell anemia patients to increase the production of fetal hemoglobin in their red blood cells (ClinicalTrials.gov: NCT03745287). Although it is too early to determine whether these therapies are successful, CRISPR-Cas9-based gene editing has begun to move out of research laboratories into clinics worldwide. Genome Editing for NF1. Despite the promise, clinical translation of the Cas9-sgRNA technology for disease treatment is contingent on the development of a delivery system that can efficiently and safely target cells (described below).36Nelson C.E. Gersbach C.A. Engineering delivery vehicles for genome editing.Annu. Rev. Chem. Biomol. Eng. 2016; 7: 637-662Crossref PubMed Scopus (82) Google Scholar While genome editing has not yet been attempted as a therapy for NF1, it would be applicable to most small mutations. 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