Abstract
Neurofibromatosis type 2 (NF2)-related schwannomatosis is a rare autosomal dominant monogenic disorder caused by mutations in the NF2 gene. The hallmarks of NF2-related schwannomatosis are bilateral vestibular schwannomas (VS). The current treatment options for NF2-related schwannomatosis, such as observation with serial imaging, surgery, radiotherapy, and pharmacotherapies, have shown limited effectiveness and serious complications. Therefore, there is a critical demand for novel effective treatments. Gene therapy, which has made significant advancements in treating genetic diseases, holds promise for the treatment of this disease. This review covers the genetic pathogenesis of NF2-related schwannomatosis, the latest progress in gene therapy strategies, current challenges, and future directions of gene therapy for NF2-related schwannomatosis.
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Neurofibromatosis type 2 (NF2)-related schwannomatosis is an inherited multiple neoplasia syndrome with an incidence of 1:25,000–1:40,000, caused by loss-of-function mutations in the NF2 gene. |
There is a critical demand for novel effective treatments for NF2-related schwannomatosis since the current treatment options have shown limited effectiveness and serious complications. |
Gene therapy which has displayed encouraging efficacy in preclinical studies holds promise for the treatment of NF2-related schwannomatosis in the future. |
This review covers the genetic pathogenesis of NF2-related schwannomatosis, the latest progress, current challenges, and future directions in gene therapy for NF2-related schwannomatosis. |
Introduction
Neurofibromatosis type 2 (NF2)-related schwannomatosis is an inherited multiple neoplasia syndrome with an incidence of 1:25,000–1:40,000, caused by loss-of-function mutations in the NF2 gene that encode the tumor suppressor merlin [1,2,3,4,5,6]. The distinctive tumor of NF2-related schwannomatosis is bilateral vestibular schwannomas (VS) (Fig. 1). Meningiomas, ependymomas, cutaneous tumors, and abnormal ocular findings are also commonly observed in these patients with NF2-related schwannomatosis [7,8,9,10,11,12,13,14,15]. The phenotypic variations of NF2-related schwannomatosis are correlated with the mutation type or location in the NF2 gene [16].
MRI imaging of bilateral vestibular schwannomas (VS) in patients with neurofibromatosis type 2 (NF2)-related schwannomatosis. The yellow arrow points to bilateral VS. a, b Bilateral VS before surgery. c, d Bilateral VS after removing a unilateral VS by surgery. M male, F female
Current treatment recommendations for NF2-related schwannomatosis include close observation with serial imaging, surgery (Fig. 1), radiosurgery, and pharmacotherapies [17, 18]. Surgery and stereotactic radiation are considered effective approaches for managing NF2-related VS. However, these treatments can also result in severe complications such as nerve injury, deafness, facial paralysis, and radiation-induced secondary malignancy [19,20,21,22,23,24,25,26,27,28]. Several clinical trials of specific pharmacotherapies are ongoing or have been completed, involving angiogenesis inhibitors (bevacizumab, axitinib, sorafenib) [29,30,31,32], receptor tyrosine kinases (RTKs) inhibitors (lapatinib, erlotinib, crizotinib, brigatinib) [33, 34], Mammalian target of rapamycin (mTOR) inhibitors (everolimus, vistusertib) [35,36,37,38,39], mitogen-activated protein kinase (MEK) inhibitors (selumetinib) [40, 41], histone deacetylase (HDAC) inhibitors (AR-42) [42, 43], proinflammatory mediator-targeting drugs (aspirin) [44,45,46,47], and peptide vaccine therapy for vascular endothelial growth factor (VEGF) receptor [48, 49]. Among these, the humanized anti-VEGF antibody bevacizumab has shown efficacy in hearing improvement and VS regression in approximately 50% of patients [29,30,31]. However, its long-term effects are limited because of dose-limiting toxicities associated with prolonged use of the drug [32]. Hence, there are unmet medical needs for effective, safe, and continuous treatments for NF2-related schwannomatosis.
Over the past years, gene therapy, especially gene replacement therapy, has made impressive progress in treating hereditary diseases, including spinal muscular atrophy, monogenic immunodeficiency diseases, Leber congenital amaurosis, and hemophilia [50,51,52,53]. Moreover, several clinical trials of gene therapy are underway for several neurologic disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Canavan disease (CD), giant axonal neuropathy (GAN), and aromatic l-amino acid decarboxylase (AADC) deficiency [54,55,56,57,58,59]. Adeno-associated virus (AAV) vectors for gene delivery in vivo are demonstrated to be efficient and safe in the nervous system. NF2-related schwannomatosis tumor is an appropriate target for gene therapy, as it grows slowly and can be easily localized and monitored using MRI. Recently, AAV-based gene replacement therapy has displayed encouraging efficacy in a preclinical schwannoma sciatic nerve xenograft mouse model of NF2-related schwannomatosis. Thus, gene therapy holds potential as an effective and promising therapeutic strategy for NF2-related schwannomatosis in the future.
New advances in molecular biology and pathogenic genetics study on NF2-related schwannomatosis help to establish novel gene replacement therapy to restore merlin expression, potentially providing an effective treatment for the disease. This review discusses the genetic pathogenesis of this disorder, current position, and future directions of gene therapy strategies for NF2-related schwannomatosis.
Ethical approval
This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.
Genetic Pathogenesis of NF2-Related Schwannomatosis
The NF2 gene is the target for NF2-related schwannomatosis gene therapy. The genetic pathogenic mechanisms of the mutated NF2 gene are the basis for designing optimal gene therapy strategies with high efficiency and low toxicity. This section introduces the NF2 gene, two-hit tumor formation, and the correlation between genotype and phenotype in NF2-related schwannomatosis.
NF2 Gene and Merlin Protein
NF2-related schwannomatosis is caused by mutations in the NF2 gene located on chromosome 22q12.2 with a total length of approximately 95 kilobases (kb) [60,61,62]. The NF2 gene, identified as a tumor suppressor gene in 1993, contains 17 coding exons and encodes a 65–70 kDa protein called merlin, which is a tumor suppressor localized to the interface between the cell membrane and cytoskeleton [63,64,65]. Of the two major isoforms (I and II) of merlin, only merlin isoform I can form an active closed conformation and possesses tumor suppressor activity [66,67,68,69,70,71,72,73,74]. Merlin is involved in various signaling pathways, including PI3K/Akt/mTORC1, Ras/Raf/MEK/ERK, receptor tyrosine kinases, and Hippo signaling, to regulate cell growth, proliferation, and survival [75,76,77,78,79,80,81,82,83,84], and control cell motility, morphology, and cell communication [85,86,87,88,89,90,91,92,93,94]. Mutations in the NF2 gene produce a nonfunctional merlin protein, which leads to uncontrolled cell proliferation and tumorigenesis.
Two Genetic Hits to NF2-Related Schwannomatosis
Tumor formation in patients with NF2-related schwannomatosis is thought to be consistent with Knudson’s two-hit hypothesis of tumorigenesis [95, 96]. Tumor occurs when both alleles of the NF2 gene are inactivated. Approximately 50% of patients with NF2-related schwannomatosis inherit a germline mutation of one allele of the NF2 gene from an affected parent. The remaining 50% of patients with NF2-related schwannomatosis with no history of the disorder in their family have a de novo mutation in one allele of the NF2 gene at the postzygotic stage of embryogenesis. This de novo mutation of the NF2 gene is a somatic mutation leading to two separate cell lineages (cells with and without the mutation) in patients and results in somatic mosaicism [97]. About 60% of patients with de novo mutation in the NF2 gene are mosaics [98], but the actual detection rate of the mutated gene is low at 25–30%, as only a portion of cells contain the mutated NF2 gene in mosaic patients with NF2-related schwannomatosis [99,100,101]. In patients with the first mutated allele of the NF2 gene, almost everyone acquires the second mutation in another allele of the NF2 gene in many cells of susceptible target organs including nervous system, eyes, and skin during their lifetime resulting in loss of heterozygosity (LOH) on chromosome 22 and development of multiple tumors of NF2-related schwannomatosis [102, 103].
Genotype–Phenotype Correlation in NF2-Related Schwannomatosis
NF2-related schwannomatosis shows nearly 100% penetrance by 60 years of age and has wide phenotypic variability. Disease severity is related to the site and types of NF2 gene mutation. The identification of specific genotype and phenotype correlations has provided insights into the clinical heterogeneity observed between different families. In general, nonsense or frameshift mutations in the NF2 gene that produce truncated and dysfunctional merlin proteins are closely connected with severe disease. In contrast, missense or non-truncating mutations are associated with mild disease [104, 105]. Splice-site mutations are also linked to variable disease severity [106]. The phenotype in patients with mosaicism was found to be different from that in the germline patients. The mosaic patients tend to have mild generalized or even localized diseases such as unilateral VS with ipsilateral tumors. The correlation between genotype and phenotype has been identified to help predict disease severity [107,108,109,110,111,112].
Gene Therapy Strategies and Challenges for NF2-Related Schwannomatosis
The history of gene therapy for NF2-related schwannomatosis goes back to the 2010s (Fig. 2). In 2010, the first preclinical NF2-related schwannomatosis gene therapy study was reported. A herpes simplex virus-1 (HSV-1) amplicon vector-based delivery of caspase-1 (interleukin-β-converting enzyme; ICE) led to regression of schwannoma in a subcutaneous xenograft mouse model [113]. In several further studies of gene therapy for NF2-related schwannomatosis from 2013 to 2022, adeno-associated viral vector serotype-1 (AAV1) was utilized to deliver ICE, the pore-forming protein Gasdermin-D, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and functional merlin causing schwannomas regression in a xenograft mouse model [114,115,116,117]. These studies support that gene therapy has the potential to provide therapeutic efficacy for NF2-related schwannomatosis.
The history of the development of gene therapy for neurofibromatosis type 2 (NF2)-related schwannomatosis. 1. Prior to 2010, no reported preclinical study on gene therapy for NF2-related schwannomatosis. 2. In 2010, HSV-based delivery of ICE led to schwannoma regression in a subcutaneous xenograft mouse model. 3. In 2013, AAV1-based delivery of ICE led to schwannoma regression in a sciatic nerve xenograft mouse model. 4. In 2019, AAV1-based delivery of the pore-forming protein Gasdermin-D led to schwannoma regression in a sciatic nerve xenograft mouse model. 5. In 2019, AAV1-based delivery of ASC led to schwannoma regression in a sciatic nerve xenograft mouse model. 6. In 2022, AAV1-based delivery of merlin protein led to schwannoma regression in a sciatic nerve xenograft mouse model. 7. In 2022, Materials and methods for neurofibromin 2/merlin (NF2) gene therapy are disclosed in the international patent application No. PCT/US2022/024680 published as WO 2022/221447A1, filed by RES INST Nationwide Children’s Hospital on April 13, 2022, which claims the priority to US provisional patent application No. 63/174,803, filed on April 14, 2021. 8. In 2023, further preclinical studies on gene therapy for NF2-related schwannomatosis are ongoing. HSV, herpes simplex virus; ICE, caspase-1; AAV1, adeno-associated viral vector serotype-1; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain
Currently, gene therapy approaches for NF2-related schwannomatosis primarily include suicide gene therapy, gene replacement or augmentation therapy, and gene knockdown and replacement combination approach (Fig. 3). Although these gene therapy strategies have shown potential in preclinical animal model studies, they still face many specific challenges apart from the traditional challenges for gene therapy such as immunogenicity, delivery vector, manufacturing, and the long-term effects of treatments (Table 1) [118].
Gene therapy approaches for neurofibromatosis type 2 (NF2)-related schwannomatosis. A Suicide gene therapy refers to the specific introduction of a gene into tumor cells to cause tumor cell death without affecting the healthy nearby cells. B Gene replacement therapy directly supplies a functional copy of the mutated or inactivated NF2 gene to augment functional merlin protein re-expression in NF2-deficient tumor cells. C Gene knockdown and replacement combination approach refers to using small RNAs to silence the mutated NF2 gene while supplying a functional copy of the NF2 gene to produce normal merlin protein
Suicide Gene Therapy and Extracellular Vesicles (EVs)
Suicide gene therapy for NF2-related schwannomatosis introduces the specific gene into tumor cells, which can cause the death of tumor cells without affecting the healthy nearby cells. Although clinical data on the application of suicide gene therapy for NF2-related schwannomatosis tumors do not exist, several preclinical animal studies have shown promising results in recent years. It was reported in 2013 that in a xenograft mouse model in which human NF2-related schwannomatosis tumors form in the distal sciatic nerve of nude mice, direct intra-tumoral injection of an AAV1 vector encoding ICE under the control of the Schwann cell (SC)-specific promoter P0 (AAV1-P0-ICE) led to selective death of tumor cells, preventing the development of early schwannomas and causing regression of well-established tumors with minimal to no nerve damage [114]. More recent studies in 2019 showed regression of NF2-related schwannomas without detectable toxicity using an AAV1 vector to deliver the pore-forming protein Gasdermin-D and ASC that induce tumor cell death under the control of the SC-specific promoter P0 in an orthotopic xenograft mouse model [115, 116]. These preclinical studies demonstrate the efficacy and safety of the suicide gene therapy for NF2-related schwannomatosis tumors and will help conduct translational research to support the transition to clinical trials. The approach may have a “bystander effect,” such that transduced schwannoma cells can inhibit the growth of non-transduced cells in the tumor by EV delivery. Moreover, this approach not only could eliminate the treated tumors but also has the potential to initiate adaptive antitumor immunity leading to regression of distal NF2-related schwannomatosis lesions. Compared with surgical resection, the advantage of suicide gene therapy involves utilizing minimally invasive approaches with minimal complications in patients with NF2-related schwannomatosis, such as direct intra-tumoral injection under MRI or ultrasound guidance. In addition, it is expected that these suicide gene therapies may be combined with surgical resection and other currently available treatments to achieve potentially synergistic and additive therapeutic effects [114,115,116].
EVs are a promising and appealing delivery vehicle for therapeutic proteins, which encompass various types of vesicles, including microvesicles, apoptotic bodies, oncosomes, and exosomes. Among the different types of EVs, exosomes and microvesicles have been extensively investigated. In a preclinical study of suicide gene therapy for NF2-related schwannomatosis, EV delivery of overexpressed ICE to treat schwannoma leading to regression of well-established tumors is an example of protein delivery for therapeutic benefit. In a xenograft mouse model in which human NF2-related schwannomatosis tumors form in the distal sciatic nerve of nude mice, direct intra-tumoral injection of an AAV1 vector encoding ICE under the control of the SC-specific promoter P0 (AAV1-P0-ICE) led to death of tumor cells and caused regression of well-established tumors. Although only approximately 20% of tumor cells were affected by the gene delivery, it is remarkable that the schwannoma tumors exhibited nearly complete regression. The study’s data provides support that EVs may play a role in the transfer of ICE protein and/or mRNA between transduced and non-transduced schwannoma cells within the tumor microenvironment, potentially contributing to the bystander killing mechanism in schwannoma [119, 120].
Gene Replacement Therapy
Gene replacement therapy for NF2-related schwannomatosis directly supplies a functional copy of the mutated or inactivated NF2 gene to augment functional merlin protein re-expression in NF2-deficient tumor cells to treat the disease phenotype caused by the defective NF2 gene. This approach is also called gene augmentation therapy, which has recently made encouraging progress in a preclinical study in 2022. In a xenograft mouse model in which human NF2-null SC-derived tumors were growing in the sciatic nerve of nude mice, a single intra-tumoral injection of an AAV1 vector directly delivered merlin gene under the control of CBA promoter (AAV1-CBA-merlin) and resulted in overall regression of tumors and no toxic effects to animals, which seems to be an unexpected therapeutic effect [117].
Another gene replacement therapy study for NF2-related schwannomatosis was disclosed in a PCT patent application published in October 2022, WO 2022/221447 A1, which relates to materials and methods for neurofibromin 2/MERLIN (NF2) gene therapy. In a xenograft mouse model in which human NF2-related schwannomatosis tumors form in the distal sciatic nerve of nude mice, direct intra-tumoral injection of an AAV9 vector encoding merlin (AAV9-NF2) stopped the growth of existing tumors, shank existing tumors, and reduced or prevented the formation of new tumors. The PCT patent also provides a gene therapy method comprising administering a gene therapy vector into the tumor or cerebrospinal fluid by direct injection into the tumor, systemic delivery routes such as intravenous delivery, intrathecal delivery, or any other delivery method for direct application of the vector. These findings bring hope for the clinical application of gene replacement therapy for NF2-related schwannomatosis.
NF2-related schwannomatosis is a monogenic genetic disease with a single known genetic mutation of the NF2 gene. Compared with the current treatments such as surgery and pharmacotherapies, the advantage of gene replacement therapy is that it can directly target the “driver” mutated NF2 gene and restore its function by delivering a functional copy of the NF2 gene to re-expression merlin protein in tumor cells. This inhibition may have a bystander effect to suppress the growth of non-transduced tumor cells by transferring overexpressed merlin in transduced tumor cells to non-transduced tumor cells via EVs [117,118,119,120]. This approach can not only treat existing tumors but may also be used as a preventative prophylactic gene therapy to inhibit future tumor formation by delivering AAV‑merlin vectors intravenously and intrathecally with access to NF2± heterozygous cells throughout the body. Therefore, the effectiveness of gene replacement therapy could be expanded by transferring merlin via EVs or by delivering AAV‑merlin vectors intravenously and intrathecally. Gene replacement therapy can also be combined with surgical resection and other currently available treatments to achieve potentially synergistic and additive therapeutic effects [117].
Gene Knockdown and Gene Replacement Combination Therapy
This combination therapy for NF2-related schwannomatosis refers to using small RNAs to silence the mutated NF2 gene while supplying a functional copy of the NF2 gene to produce normal merlin protein. Gene silencing therapies using small RNAs aim to intervene in gene transcription and translation of the mutated NF2 gene. In theory, gene knockdown and gene replacement combination therapy addresses the problem of whether the existing mutated NF2 gene has a dominant negative effect on the cell [118, 121,122,123,124,125,126]. The study on this treatment approach is ongoing.
Gene Delivery Strategy and Challenges for NF2-Related Schwannomatosis
Gene delivery strategy involves vector selection, vector delivery approaches, and vector production (Table 2). The gene therapy vectors, which are the key to the success of gene therapy, include viral vectors and non-viral vectors. The design of an optimal gene delivery system depends on several factors, including the target cells, the size of the gene, the immunogenicity and specificity of the vector, the duration of expression, the optimal route of administration, and the vector manufacturing complications [127, 128].
Viral Vector Selection and Delivery
AAV is the most widely used vector for gene therapy in clinical practice. So far, various gene therapy drugs using AAV as a vector have been approved for marketing. When considering the vector selection for NF2-related schwannomatosis gene therapy, AAV is a wise choice for many reasons, including low immunogenicity, tissue-specific tropism, high efficiency and specificity in transduction, long-term stability, no incorporation into the host chromosome, low genotoxicity, safety, and existing manufacturing expertise. There are 13 common AAV serotypes or variants (AAV1–13) identified to date, providing multiple options [129,130,131,132,133,134,135,136,137]. In the preclinical study of NF2-related schwannomatosis gene therapy, the most preferred and studied AAV serotypes are AAV1 and AAV9. AAV9 is suitable to be utilized to develop a novel gene therapy product without concern regarding ownership of the intellectual property (IP) since the use of AAV9 can be acquired through purchase [113,114,115,116,117,118,119]. The dual-AAV systems have proven effective in transducing gene sequences of larger sizes [138]. The findings of these studies will contribute to enhancing the safety and effectiveness of AAV-NF2 delivery in clinical trials. In addition, non-viral vectors such as liposomes and peptide-based nanoparticles, although less commonly used than AAV and other viral vectors, offer an appealing option for the delivery of the NF2 gene in the preclinical study of NF2-related schwannomatosis gene therapy [139, 140].
The gene therapy vector must be efficiently delivered to affected cells in patients to achieve functional expression of targeted genes in specific tissues to treat the disease. For NF2-related schwannomatosis gene therapy, AAV therapeutic vectors can be delivered by direct intra-tumoral injection, intravenous (IV) injection, intrathecal injection, intraventricular injection, and intra-arterial (IA) injection [117, 118]. The method of vector delivery will depend on the approach of gene therapy and the desired therapeutic outcome; that is, the direct intra-tumoral injection will exhibit superior efficacy in the eradication of tumors by suicide gene therapy, whereas intravenous injection or intrathecal injection will be more beneficial to inhibit tumors growth throughout the body. Considering that the majority of NF2-related schwannomatosis tumors are situated in anatomically inaccessible locations such as the brain and spine, the administration of the viral vectors into the cerebrospinal fluid (CSF) may provide a more effective pathway to reach specific tumors in patients. IA injection delivery may also be taken into consideration. However, the clinical experience with this method for administering chemotherapy drugs to treat brain tumors has demonstrated minimal or negligible advancements compared to IV injection administration. Since most NF2-related schwannomatosis tumors are considered solid tumors, how to ensure the penetrance of gene therapy drugs if delivered systemically is a big challenge. In the preclinical animal models studied for NF2-related schwannomatosis gene therapy, AAV therapeutic vectors are delivered by direct intra-tumoral, IV, intraventricular, and intrathecal injection, thereby stopping the growth of existing tumors [113,114,115,116,117,118,119]. Despite ongoing efforts, there are still challenges in the development of clinically applicable methods to deliver gene therapy vectors to specific tumor locations in individuals with NF2-related schwannomatosis. By refining the injection technique through the round-window membrane, it is possible to optimize the drug’s pharmacokinetics and pharmacodynamics within the inner ear. The efficacy and safety of these vector delivery approaches for NF2-related schwannomatosis gene therapy must be thoroughly studied [140,141,142].
Viral Vector Production
Several manufacturing methods have been established for producing AAV vectors. One commonly used method is the transient transfection method in HEK293 cells, with the advantage of producing new AAV constructs quickly without residual helper virus contaminants. However, disadvantages of this production method are its often lower yields and efficiency and difficultly in scaling up from the bench to a commercial size [143,144,145,146,147]. To address the challenges in the manufacture of gene therapy products, HEK293 cells have been adapted for suspension growth to improve production yields and facilitate scale-up [148]. The development trend of AAV vector production is to use stable transfection and suspension culture to reduce costs and expand production capacity. The manufacturing of viral vectors for NF2-related schwannomatosis gene therapy faces many technological, facility, and expertise barriers. Current difficulties in viral vector production include reducing the number of plasmids required for transient transfection, improving transfection efficiency, increasing cell culture density, expanding production capacity, and reducing the empty shell rate. It is necessary to consider the selected viral vector’s production scale and large-scale manufacturing protocols. The large-scale manufacturing of viral vectors is facing challenges of being cost-effective at a commercial scale. Contract Development and Manufacturing Organization (CDMO) is an essential participant in the industrial chain of gene therapy and can solve the large-scale manufacturing bottleneck of viral vectors for NF2-related schwannomatosis gene therapy [118, 146].
Preclinical Models for NF2-Related Schwannomatosis Gene Therapy
Cell models and animal models of NF2-related schwannomatosis are the basis for the study of this rare disease. The development of gene therapy for this disease has been greatly impeded by the absence of suitable cell models and animal models. Therefore, novel clinically relevant NF2-related schwannomatosis models are urgently needed for gene therapy research [142]. Advances in genome-editing methods and organoid technology have opened new avenues for the establishment of preclinical models for NF2-related schwannomatosis gene therapy.
Cell Models
The commonly used cell model of NF2-related schwannomatosis schwannoma is the HEI-193 cell line, which was initially immortalized from the tumor acquired from a patient with bilateral VS. The mutation present in this cell line results in a splicing defect within the NF2 gene, leading to the expression of a specific merlin isoform. However, HEI-193 cells can not accurately phenocopy the NF2-related schwannomatosis schwannoma cell types because they exhibit aggressive growth [142, 149]. In the preclinical studies of gene replacement therapy for NF2-related schwannomatosis by Dr. Breakefield and colleagues, clustered regulatory interspaced palindromic repeats (CRISPR)-edited human NF2-deficient immortalized arachnoid cells (AC) or SCs were established and used as cell models [117]. Recently, the establishment of a patient-derived cell model of NF2-related schwannomatosis schwannoma was reported by Dr. Zhao. The model closely replicates genotypes and phenotypes observed in patients’ tumors. This cell model may be a reliable preclinical tool for gene therapy study [150]. Although there are several cell models available, development of more novel cell models that can accurately replicate the characteristics of NF2-related schwannomatosis tumor cells is needed. It may potentially provide insights into NF2 gene variation to create induced pluripotent stem cell lines (iPSC) as a method to establish patient-derived NF2-related schwannomatosis schwannoma cell lines [118, 142].
Animal Models
An effective preclinical animal model of human NF2-related schwannomatosis should contain extensive physiological relevance at both the genotypic and phenotypic levels [118]. In the current preclinical studies of gene therapy for NF2-related schwannomatosis, the utilized xenograft mouse models were HEI-193 cell-derived tumors or the CRISPR-edited NF2-null human SC-derived schwannomas formed in nude mice sciatic nerve [114,115,116,117, 151, 152]. Other schwannoma mouse models can also be used in future tests, such as a mouse SC4 schwannoma cell-derived allograft schwannoma model [153], a genetically engineered mouse (GEM) model (Periostin-Cre NF2flox/flox mouse) [154], and a patient-derived xenograft (PDX) mouse model [150]. However, most existing mouse models of NF2-related schwannomatosis do not display the occurrence of VS. Although the current genetically engineered mouse models of NF2-related schwannomatosis are beneficial for studying disease mechanisms, it remains unclear if this model can be regarded as an accurate mouse model for gene augmentation therapy of the disease [154, 155, 156]. It is difficult to establish PDX models for NF2-related schwannomatosis because of the slower growth and lack of transplantation capability exhibited by these xenografts. Recently, Dr. Zhao reported the establishment of PDX models of NF2-related schwannomatosis schwannoma, which may be a reliable preclinical tool for gene therapy study [150, 157, 158]. It is important to evaluate the efficacy of gene therapy drugs in non-human primate (NHP) animal models. However, it will take a long time and high cost to develop the NHP models. In addition, one needs to assess the immune response to viral vector-based treatment and the use of immunosuppressants in immune-competent animal models [118].
Future Directions
Gene Editing
Gene editing technology is a technique that enables precise modifications to the genome using engineered nucleases. It has emerged as an ideal platform for knocking out/in and replacing specific DNA fragments to perform accurate gene editing on the genome level. By precisely modifying the genetic mutations, this technology holds great promise for treating various genetic disorders. In the development history of gene editing techniques, the discovery of the CRISPR-associated protein 9 system (CRISPR/Cas9) in 2012 is a milestone. The improvement of CRISPR/Cas9 gene editing has spurred the progress of base editing and prime editing (PE), which can enable the precise alteration or elimination of a mutation linked to specific diseases [159,160,161,162,163,164,165,166]. On December 9, 2023, the US Food and Drug Administration (FDA) approved the Casgevy therapy for market use in treating sickle cell disease, making it the first CRISPR gene editing therapy approved by the FDA. Other clinical trials of CRISPR gene editing therapy for disease treatment are ongoing. For example, EDIT-101 (developed by Editas Medicine, NCT03872479) is aimed at addressing Leber congenital amaurosis type 10 (LCA type 10), using CRISPR/Cas9 and prime editing [167, 168]. Although gene editing technology was not reported to be utilized in preclinical studies of NF2-related schwannomatosis, these new technologies are innovative and promising new treatment tools for this disease in the future.
Non-Viral Vectors
Optimal vector selection is a crucial factor in the successful implementation of gene therapy. Most recent clinical studies select to utilize viral vectors, which are generally considered safe for patients [169, 170]. However, many scientists in the field are exploring non-viral alternatives because of the potential risks associated with viral delivery systems, such as immunogenic responses and insertional mutagenesis [171, 172]. In recent years, non-viral vector systems have emerged as a rapidly evolving research area in the field of gene delivery. Compared with viral vectors, non-viral vectors are lower cost, easy to produce, and low in immunogenicity, cytotoxicity, and mutagenesis. These advantages of non-viral vectors make them attractive to researchers in advancing the field of gene therapy. However, non-viral vectors still face critical challenges, including specificity, safety, and gene transfer efficiency [171,172,173]. Before significant results of non-viral vector systems can be achieved, further improvement of the transfection efficiency is necessary. Currently, nanoparticle delivery systems are a hot research field of non-viral vectors. In the preclinical study of genetic deafness, peptide-based nanoparticles and liposomes have been employed for the delivery of genetic materials [139, 140]. The non-viral vector presents a hopeful alternative for future research on gene delivery in the treatment of this disease.
Biomarker Identification
The identification of molecular biomarkers in evaluating the efficacy of gene therapy for NF2-related schwannomatosis will improve the efficiency of gene therapy transfer from “bench to bedside”. Therefore, it is necessary to identify more effective molecular biomarkers compared to MRI imaging. Ki‑67 and MIB‑2, two histological biomarkers of cell proliferation, have been found to correlate with tumor growth in patients with VS [174]. In the preclinical study of NF2-related schwannomatosis gene replacement therapy by Dr. Breakefield and colleagues, Ki-67 was used as a biomarker to show the inhibition of cell proliferation [117]. Unfortunately, it is not feasible to perform nondestructive biopsies on the inner ear tissue. Considering practicality and invasiveness in evaluating the efficacy of gene therapy, a serum biomarker would be ideal compared to MRI imaging. Currently, no known blood-borne biomarker for NF2-related schwannomatosis can be monitored. The peripheral blood concentration of VEGF and the mean apparent diffusion coefficient of radiographic factors may function as biomarkers to predict which individuals with NF2-related schwannomatosis are likely to experience significant benefits from anti-VEGF therapy [175,176,177,178]. However, it is not clear whether they can be employed to evaluate the efficacy of NF2-related schwannomatosis gene therapy. CSF is a source of potential biomarkers for NF2-related schwannomatosis. To date, the significance of the correlation between disease severity and CSF composition remains unreported [178, 179]. Given that normal cells and tumor cells do not typically secrete merlin, it is imperative to determine the feasibility of utilizing western blot to detect tumor merlin levels to track tumor progression. Developing more convenient, practical, and less invasive ways than performing routine MRI scanning to determine merlin expression in patients with NF2-related schwannomatosis may address the problem [118]. Within the cochlea, proteins secreted by the inner ear cells are enriched in perilymph fluid. Several studies have indicated the potential for the discovery of biomarkers associated with NF2-related schwannomatosis from liquid biopsy of human perilymph [180, 181]. In the future, the discovery and validation of molecular biomarkers could be more efficient by using “omics” technologies such as epigenetic studies, high-throughput proteomics, and genome-wide screening [142, 182].
Conclusion
Gene therapy is a promising new treatment for single-gene genetic diseases with definite causative genes, which provides patients with the hope of a one-time permanent cure. Rapid progress in current preclinical studies has shown that NF2-related schwannomatosis gene therapy not only can be used to treat existing tumors but may also be used as a preventative prophylactic approach to inhibit future tumor formation and may be combined with surgical resection and other currently available treatments to achieve potentially synergistic and additive therapeutic effects. Because of the limitations of the existing treatment approach and the lack of safe, effective, and durable therapies, gene therapy is expected to become the necessary fundamentally new treatment for NF2-related schwannomatosis with minimal complications and side effects in the future. Although there are still many challenges, with gene therapy’s rapid and iterative development from science and technology to innovation, clinical applications will continue to make breakthroughs. The gene therapy for NF2-related schwannomatosis is moving to the clinic with a promising future.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Minjun Yan, Yu Zhang, Hanlu Tang, and Zhixu Bie for contributing to the manuscript preparation.
Funding
This study was funded by grants from the National Natural Science Foundation of China (grant numbers 82373451 and 81974387), the Beijing Municipal Natural Science Foundation (No. L222015), the Platform Construction of Basic Research and Clinical Translation of Nervous System Injury (2020) (number PXM2020_026280_000002). The Rapid Service Fee was funded by the authors.
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Conception and design: Ruofei Yuan, Bo Wang, Ying Wang, Pinan Liu; Writing—original draft preparation: Ruofei Yuan; Writing – review and editing: Bo Wang, Ying Wang, Pinan Liu; Funding acquisition: Bo Wang, Pinan Liu; Supervision: Pinan Liu.
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Ruofei Yuan, Bo Wang, Ying Wang, and Pinan Liu declare that they have no competing interests.
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This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.
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Yuan, R., Wang, B., Wang, Y. et al. Gene Therapy for Neurofibromatosis Type 2-Related Schwannomatosis: Recent Progress, Challenges, and Future Directions. Oncol Ther 12, 257–276 (2024). https://doi.org/10.1007/s40487-024-00279-2
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DOI: https://doi.org/10.1007/s40487-024-00279-2