# Advancing Neurofibromatosis Type 1 Therapies through
Basic Research Translation

Jude Oluwapelumi Alao, Oluwaseyi Joy Alao, Zainab Precious Adebayo

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Jude Oluwapelumi Alao, Oluwaseyi Joy Alao, Zainab Precious Adebayo. Advancing Neurofibromatosis Type 1 Therapies through Basic Research Translation. International Research Journal of Oncology, 2023, 6 (2), pp.156-167. hal-04184326v2

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# Advancing Neurofibromatosis Type 1 Therapies through Basic Research Translation

Jude Oluwapelumi Alao<sup>a*</sup>, Oluwaseyi Joy Alao<sup>b</sup> and Zainab Precious Adebayo<sup>a</sup>

## Authors' contributions

This work was carried out in collaboration among all authors. Author AJO designed the study and wrote the first draft of the manuscript. Authors AOJ and AZP managed the literature searches. All authors read and approved the final manuscript.

## Article Information

### Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/104508

## Review Article

Received: 05/06/2023

Accepted: 11/08/2023

Published: 21/08/2023

## ABSTRACT

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by the growth of both benign and malignant tumors in neural crest cells. Despite extensive research, only one medication has been approved for plexiform neurofibroma treatment, and no specific drugs exist for other associated tumors. Recent studies suggest targeting cellular signaling pathways (Hippo, Janus kinase/signal transducer and activator of transcription, and mitogen-activated protein kinase) and the microenvironment (nerve cells, macrophages, mast cells, and T cells) as potential therapeutic approaches. Several clinical trials are investigating agents that inhibit specific kinases or target signaling molecules in the microenvironment. Despite significant progress, more effective treatments are needed. This paper reviews previous strategies, ongoing clinical trials, and recent advances in basic research related to NF1 and its associated tumor. Data collected from scientific databases and literature highlight the potential of novel therapeutics, including kinase inhibitors,

<footnote><sup>a</sup> Microbiology Department, Adeleke University, Ede, Osun State, Nigeria.</footnote>

<footnote><sup>b</sup> Herefordshire and Worstershire Health and Care NHS Trust, UK.</footnote>

<footnote>*Corresponding author: E-mail: alaojude@adelekeuniversity.edu.ng;</footnote>
signaling molecule modulators, and microenvironment-targeting drugs. Understanding the underlying mechanisms of tumor development and progression in NF1 is crucial for developing effective therapies. This review provides insights into current strategies, ongoing trials, and recent basic research, aiming to improve treatment outcomes and address unmet needs in NF1 management. By bridging the gap between basic research and clinical translation, this work paves the way for advancing NF1 therapies and bringing novel treatments to patients.

Keywords: NF1; tumor; signaling; therapies; cell.

## 1. INTRODUCTION

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve and skin tumor. Despite its high prevalence, effective treatments for NF1 have been limited due to a lack of understanding of the disease biology. However, recent advances in NF1 research have opened doors for therapeutic development. Key progress has been made through basic research translation, applying findings to treatment development. Improved genetic techniques and mouse models have enhanced our understanding of NF1's biology. Human induced pluripotent stem cells (iPSCs) have also provided valuable insights [1].

NF1 research has revealed dysregulated RAS/MAPK signaling as a critical factor in tumor development. Mouse models with NF1 mutations have been crucial in studying disease biology and identifying potential therapies. Inhibiting the RAS/MAPK pathway has shown promise in reducing tumor growth. Using iPSCs has furthered our understanding of NF1, enabling the study of NF1 gene mutations and testing novel treatments. These advancements offer new prospects for NF1 therapy development. Basic research translation is a crucial link between research findings and treatment innovation. The combination of improved genetic techniques, mouse models, and iPSC research has paved the way for deeper insights into NF1 biology and exploring new therapies. The ultimate goal is to develop effective treatments for patients with NF1.

## 2. MAPPING THE LANDSCAPE OF NEUROFIBROMATOSIS: A HISTORICAL OVERVIEW

The condition was first described in the medical literature over a century ago and has since been the subject of much research and investigation.

The first description of a condition now known as NF1 can be traced back to the late 19th century. In 1882, German physician Friedrich Daniel von

Recklinghausen first described the condition as a "disorder of the skin and peripheral nerves" characterized by the growth of tumors on nerves and the presence of café-au-lait spots [2]. This description marked the first recognition of a condition that would come to be known as NF1. The recognition of NF1 as a distinct disorder marked a significant milestone in the understanding of the disease. However, it was not until the late 20th century that the genetic basis of NF1 was discovered. The condition was later described in detail by several other physicians, including French neurologist Paul Marie Louis Pierre Broca, who described a similar condition in 1886 [3]. These early descriptions laid the foundation for further study and investigation into the condition and helped to establish NF1 as a distinct disorder.

During the early 20th century, advances in medical science and technology led to a greater understanding of NF1 and the underlying genetic causes of the condition. In 1910, American physician George Pack reported the first case of NF1 in the United States [4]. In 1987, the genetic basis of NF1 was discovered when the gene responsible for the condition was identified and located on chromosome 17 [5]. The discovery of the genetic basis of NF1 was made possible by advances in genetic technology and the study of families with multiple cases of the disease. In 1987, the NF1 gene was identified and cloned, marking a major step forward in understanding the disease [6]. Identifying the NF1 gene allowed researchers to study the disease's underlying biology and develop new treatments. Over the past few decades, there have been many advances in our understanding of NF1 and its underlying mechanisms. In 2003, the complete sequencing of the human genome allowed for the identification of several other genes associated with NF1 [7]. This discovery has helped to shed light on the complex genetic basis of the condition and has provided new avenues for research and investigation.

There has been growing interest in developing personalized therapies for NF1 in recent years.
With the advent of new technologies and techniques, researchers can now study the disease at a molecular level and develop new treatments that target the underlying genetic causes of the condition [8]. This research holds great promise for those affected by NF1, as it offers the potential for more effective treatments and improved quality of life.

The history of NF1 is a testament to the progress made in our understanding of the condition over the past century. From the initial descriptions of the disorder by von Recklinghausen and Broca to the current advances in our understanding of the underlying genetic causes of NF1, the history of this condition is marked by a steady progression of knowledge and understanding. With continued research and investigation, our understanding of NF1 will likely continue to grow, leading to the development of new treatments and improved outcomes for those affected by this debilitating condition.

### 3. CLINICAL MANIFESTATIONS OF NEUROFIBROMATOSIS TYPE 1

Neurofibromatosis type 1 is a genetic disorder that affects multiple systems in the body, including the nervous system, skin, bones, and eyes. The clinical features of nf1 can vary widely from person to person, but there are some common manifestations of the condition (Table 1).

One of the hallmark features of nf1 is the presence of café-au-lait macules (Fig. 1), which are flat, light brown patches on the skin. These macules are present in up to 99% of people with nf1, and their number and size increase with age [9,10]. The presence of six or more café-au-lait macules that are larger than 5 mm in diameter is a diagnostic criterion for NF1 [11].

Another common feature of NF1 is the development of neurofibromas (Fig. 2), benign tumors arising from Schwann cells in the peripheral nervous system [12]. Neurofibromas can be classified into two types: cutaneous neurofibromas and plexiform neurofibromas. Cutaneous neurofibromas are small, flesh-colored bumps on or under the skin, while plexiform neurofibromas are larger and involve multiple nerves [13].

In addition to café-au-lait macules and neurofibromas, people with NF1 may also develop other skin findings, such as freckling in

the axillary or inguinal regions and skin-fold freckling [13,14]. Skeletal abnormalities, including scoliosis and bone dysplasia, are also common in NF1 [15]. NF1 can also affect the eyes (Fig. 3), with up to 50% of people with NF1 developing optic pathway gliomas. Other eye findings that can occur in NF1 include Lisch nodules, which are benign tumor on the iris, and strabismus (misaligned eyes) [16].

Finally, NF1 can cause neurological problems, such as learning disabilities, attention deficit hyperactivity disorder (ADHD), seizures, and peripheral neuropathy [16-18].

### 4. THE PAST: RETROSPECTIVE EXAMINATION OF PAST THERAPEUTIC STRATEGIES

The NF1 genetic etiology, the cellular function of the neurofibromin protein, and the identification of cellular elements that contribute to the tumor that grows in NF1 are all areas where neurofibromatosis research has made significant strides. Clinical studies of potential therapeutic targets have been conducted using this knowledge to create novel NF1 therapies (Fig. 4).

#### 4.1 Targeting Receptor Tyrosine Kinases: Inhibitors Upstream of Ras

The Ras signaling pathway is a key regulator of cell proliferation, differentiation, and survival. Aberrant activation of Ras signaling is implicated in the development and progression of many types of cancer. Ras proteins are activated by receptor tyrosine kinases (RTKs), which are frequently overexpressed or mutated in cancer cells. Therefore, targeting RTKs is a promising therapeutic approach for inhibiting Ras signaling.

RTK inhibitors are a class of drugs that target the extracellular or intracellular domains of RTKs to prevent their activation or downstream signaling. Several RTK inhibitors have been developed and tested in preclinical and clinical studies for cancer treatment. Here, we discuss some of the most promising RTK inhibitors that target the upstream of Ras.

One of the most extensively studied RTK inhibitors is erlotinib, which targets the epidermal growth factor receptor (EGFR). Erlotinib has been approved by the US Food and Drug Administration (FDA) for the treatment of non-small cell lung cancer (NSCLC) and pancreatic cancer [20]. In preclinical studies, erlotinib has
<table><caption>Table 1. Types of tumors that individuals with NF1 have a lifetime risk of developing [19]</caption><thead><tr><td>Tumor type</td><td>Lifetime risk</td></tr></thead><tbody><tr><td>Glioma of the optic pathway</td><td>15-20%</td></tr><tr><td>Other brain tumors</td><td>More than fivefold increase</td></tr><tr><td>Malignant peripheral nerve-sheath tumor</td><td>8-13%</td></tr><tr><td>Gastrointestinal stromal tumor</td><td>4-25%</td></tr><tr><td>Breast cancer</td><td>About fivefold increase</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>Fig. 1. Multiple (n ≥ 6) café-au-lait macules in a 9-year-old boy with NF1 [18]</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2a. A 46-year-old man with NF1 with numerous neurofibromas with a maximum diameter of 4 cm on his back and gluteal area. 2b. A 51-year-old lady with NF1 a tumor in her gluteal area that is 6 cm in diameter and is incapacitating [18]</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. Lisch nodules: Lisch nodules are clusters of dendritic melanocytes that appear as raised papules with a round shape and brown-yellow coloration when examined with a slit lamp [18]</figcaption></figure>

They are typically found in the iris of patients with Neurofibromatosis type 1, which is present in over 90% of cases, along with other symptoms such as café au lait pigmented skin lesions and superficial or deep neurofibromas in the skin or other organs

<figure><img src="image_5.png" /><figcaption>Fig. 4. Overview of the treatments that have been tried in the past for the most prevalent cancers linked to NF1 [19]</figcaption></figure>

The farnesyltransferase inhibitor ipifarnib, RTK inhibitors of the pathway upstream of RAS (imatinib, sunitinib, sorafenib) and mTOR inhibitors (sirolimus, everolimus)
been shown to inhibit Ras signaling and suppress tumor growth in various types of cancer [21].

"Another RTK inhibitor that has shown promising results in preclinical and clinical studies is lapatinib, which targets both EGFR and HER2 (human epidermal growth factor receptor 2)" [22-24]. "Lapatinib has been approved by the FDA for treating HER2-positive breast cancer" [23]. In preclinical studies, lapatinib has been shown to inhibit Ras signaling and induce apoptosis in cancer cells [24].

"Sorafenib is another RTK inhibitor that the FDA has approved for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma. Sorafenib targets multiple RTKs, including VEGFR (vascular endothelial growth factor receptor) and PDGFR (platelet-derived growth factor receptor), which are frequently overexpressed in cancer cells. Sorafenib has been shown to inhibit Ras signaling and induce apoptosis in cancer cells" [25].

In addition to these drugs, several other RTK inhibitors, such as gefitinib, sunitinib, and imatinib, have been approved or are in clinical development for cancer treatment [19]. While these drugs have shown efficacy in clinical trials, their efficacy can be limited by the development of resistance or toxicity.

## 4.2 Targeting the Downstream of Ras: The Promising Role of mTOR Inhibitors in NF1 Therapy

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder caused by mutations in the NF1 gene. This gene encodes for the protein neurofibromin, which functions as a tumor suppressor and regulates several signaling pathways, including the Ras signaling pathway. Dysregulation of the Ras pathway is a hallmark feature of NF1, and activation of this pathway is believed to contribute to the development of tumors in patients with NF1.

The Ras signaling pathway plays an important role in regulating cell growth and proliferation, and dysregulation of this pathway can lead to the development of various types of cancer. The mammalian target of rapamycin (mTOR) is a key downstream effector of the Ras pathway. The mammalian target of rapamycin (mTOR) is a downstream effector of the Ras pathway that plays a critical role in cell growth and proliferation. Inhibition of the mTOR pathway has

emerged as a potential therapeutic strategy for the treatment of NF1-associated tumors. mTOR inhibitors, such as rapamycin and its analogy, have shown promising results in preclinical studies and early-phase clinical trials [26].

Several studies have investigated the use of mTOR inhibitors to treat various types of NF1-associated tumors. For example, studies have shown that the mTOR inhibitor everolimus effectively reduces the size of plexiform neurofibromas, a type of tumor commonly associated with NF1, in preclinical models and early-phase clinical trials. Other studies have demonstrated the effectiveness of mTOR inhibitors in treating optic pathway gliomas, which are benign tumors that can cause vision loss in patients with NF1 [27].

mTOR is a serine/threonine kinase that forms two distinct protein complexes, mTORC1 and mTORC2, which are involved in different cellular processes. The mTORC1 complex plays a central role in regulating protein synthesis, cell growth, and autophagy, while the mTORC2 complex regulates cell survival, proliferation, and metabolism. The mTOR pathway is activated by various upstream signaling molecules, including Ras, and is frequently dysregulated in cancer cells.

The mTOR pathway can be targeted by inhibitors such as rapamycin, which inhibits mTORC1 signaling by binding to a cytoplasmic protein called FKBP12 and then binding to and inhibiting mTORC1. Rapamycin and its analogs, collectively known as rapalogs, have been shown to be effective in treating various cancer types, including neurofibromatosis, renal cell carcinoma and breast cancer.

In addition to rapamycin and its analogs, several other mTOR inhibitors have been developed that target different components of the mTOR signaling pathway. For example, the ATP-competitive mTOR inhibitor, Torin1, inhibits both mTORC1 and mTORC2 signaling by directly binding to the ATP-binding site of mTOR. Other mTOR inhibitors include AZD8055 and INK128, which inhibit both mTORC1 and mTORC2 signaling [28].

# 5. THE PRESENT: EMERGING THERAPEUTIC APPROACHES FOR THE TREATMENT OF NF1

The availability of mice models for NF1 has created previously unheard-of chances to gain
crucial insights into the biology of the illness and to establish preclinical models that direct the creation of successful treatments for NF1 (Fig. 5).

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Overview of the current treatments being researched for the most prevalent cancers linked to NF1, including immune-checkpoint inhibitors (ipilimumab, nivolumab, pembrolizumab), inhibitors of receptor tyrosine kinases (RTKs) upstream of RAS (capmatinib), components of the pathway downstream of RAS (MEK inhibitors), and BET inhibitors [19]</figcaption></figure>

**5.1 The Role of MEK Inhibition in Managing Symptomatic and Progressive Tumors in NF1**

Mitogen-activated protein kinase kinase (MEK) inhibitors are a promising therapeutic approach for NF1-related tumors, which are characterized by hyperactivation of the extracellular-signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway downstream of Ras activation. In a mouse model of NF1, MEK inhibition effectively controlled tumor size. Unlike other therapeutic strategies tested so far, MEK inhibition using selumetinib has yielded unprecedented results in the clinic for peripheral neurofibromas (pNF) [29]. "Almost all selumetinib-treated patients showed no tumor progression while being treated with the drug, with 72% showing a partial response and 24% showing stable disease in the SPRINT trial. Based on these promising results, the FDA recently approved selumetinib for children with symptomatic, progressive, and inoperable pNFs.

While MEK inhibition has also shown promise in a small cohort of children with optic pathway gliomas (OPG), equivalent strategies in the malignant context of malignant peripheral nerve sheath tumors (MPNST) have so far shown only moderate efficacy in mouse models" [30, 31]. However, it has been reported that the RTK MET is activated in some MPNSTs and that a mouse model recapitulating MET activation in MPNST is sensitive to MET inhibition. Therefore, testing a variety of MEK combination therapies, such as dual MET-MEK inhibition, in the context of MPNSTs would be interesting.

**5.2 T-Cell Therapy**

The discovery of checkpoint strategies that enable cancer to evade the cytotoxic effects of T cells has the potential to revolutionize cancer therapeutics. Despite the clinical success of immune checkpoint inhibitors, T cells have received little attention in the context of NF1. Immune profiling of neurofibromas and MPNSTs has revealed significant variation in the extent of CD8+ T cell infiltration as well as in the expression level of the T-cell inhibitory ligand PD-L1 [32,33], with 50% of gliomas containing a high number of T cells. Although the role of T cells in NF1-related tumorigenesis has not been directly evaluated, they are emerging as essential components of the tumor stroma that support tumor growth [33].

"Clinical trials targeting T cells using a combination of antibodies against the receptor for PD-L1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are currently ongoing in a Phase 2 trial for patients with rare tumors, including NF1-related tumors. However, reliable markers predicting the response to immunotherapies remain to be identified in NF1 preclinical models" [33, 34].

"Immunocompromised (T-cell-deficient) mouse models are commonly used in cancer biology to perform xenograft studies. Interestingly, malignant glioma cells successfully graft in immunocompromised mice whereas benign glioma cells fail to engraft" [35]. "This failure is due to impaired microglia function, including reduced expression of the chemokine receptor Ccr2 and its ligand Ccl5 in the immunocompromised mice. Mice deficient in Ccl5 phenocopy T-cell-deficiency, as gliomas also fail to engraft in Ccl5 knockout mice. T-cell exposure restores Ccr2 and Ccl5 expression in athymic microglia" [35]. These observations
suggest that T-cells support benign glioma
growth but impair malignant transformation. A
similar mechanism may occur in humans, as
human MPNST cells can be grown in athymic
mice [36] but a xenograft model with human
neurofibroma cells has not been reported.
Therefore, dampening T-cell function can restrain
benign tumor development while enhancing T-
cell function can be used to prevent malignant
transformation.

# 6. THE FUTURE: THE CUTTING-EDGE OF BASIC RESEARCH – UNLEASHING NEW POTENTIALS FOR THERAPEUTIC INTERVENTION

## 6.1 Gene Therapy

Gene therapy has emerged as a promising
strategy for restoring normal function of the NF1
gene. Gene therapy involves the introduction of a
functional copy of the gene into the affected cells
to restore normal protein expression and
function. Several preclinical studies have
explored the potential of gene therapy for NF1,
with promising results.

One approach to gene therapy for NF1 involves the use of viral vectors to deliver a functional copy of the NF1 gene to the affected cells. Adeno-associated virus (AAV) vectors are commonly used for gene therapy due to their safety profile and ability to integrate into the host genome. In a preclinical study, AAV-mediated delivery of the NF1 gene to Schwann cells in a mouse model of NF1 resulted in a significant reduction in tumor burden and improved survival compared to untreated mice [37]. Another study demonstrated the feasibility of AAV-mediated gene therapy in non-human primates with NF1-related optic gliomas, showing long-term transgene expression and reduced tumor size [37, 38].

In addition to AAV vectors, lentiviral vectors have also been explored for NF1 gene therapy. Lentiviral vectors have a larger capacity for gene transfer and can integrate into the host genome, allowing for stable and long-term expression of the therapeutic gene. In a preclinical study, lentiviral-mediated delivery of the NF1 gene to Schwann cells in a mouse model of NF1 resulted in a significant reduction in tumor burden and improved survival compared to untreated mice [38, 39].

While gene therapy holds great promise for
treating NF1-related tumors, several challenges

must be addressed before it can be widely
adopted in the clinic. One major challenge is the
delivery of the therapeutic gene to the affected
cells safely and efficiently. Another challenge is
ensuring long-term expression of the therapeutic
gene without inducing an immune response.
However, advances in vector technology and
gene editing techniques have brought us closer
to realizing the potential of gene therapy for
treating NF1.

## 6.2 Neuron Therapy

One promising avenue of treatment is nerve cell
therapy, which involves the transplantation of
healthy nerve cells to replace damaged or
dysfunctional cells in the nervous system.

One potential application of nerve cell therapy in NF1 is the treatment of plexiform neurofibromas (PNs), which are benign tumors that can cause significant morbidity in NF1 patients. These tumors are composed of a variety of cell types, including Schwann cells, fibroblasts, and mast cells. Previous studies have suggested that Schwann cells may play a critical role in the growth and maintenance of PNs [40]. Therefore, one approach to nerve cell therapy in NF1 is to transplant healthy Schwann cells to replace damaged or dysfunctional cells within PNs.

In recent years, there has been growing interest
in the use of induced pluripotent stem cells
(IPSCs) for nerve cell therapy in NF1. iPSCs are
generated by reprogramming adult cells, such as
skin cells, to a pluripotent state, where they can
differentiate into any cell type in the body,
including Schwann cells [40, 41]. This approach
has several advantages over traditional nerve
cell transplantation methods, including
generating an unlimited supply of cells and
avoiding immune rejection.

Several studies have investigated the potential of iPSC-derived Schwann cells for the treatment of PNs in NF1. In one study, iPSC-derived Schwann cells were transplanted into mice with NF1-associated PNs, significantly reducing tumour size and improving nerve function [42]. The transplanted cells were able to integrate into the existing nerve tissue and differentiate into functional Schwann cells. In another study, iPSC-derived Schwann cells were transplanted into rats with sciatic nerve injuries, significantly improving nerve function and regeneration [43].

While nerve cell therapy holds promise as a
potential treatment for NF1, several challenges
still need to be overcome. One major challenge is the development of safe and effective methods for delivering the transplanted cells to the affected areas of the nervous system. Another challenge is the potential for the transplanted cells to undergo malignant transformation and form tumors. Despite these challenges, the potential benefits of nerve cell therapy for treating NF1 make it a promising avenue for future research.

## 6.3 CRISPR-based Therapeutic Approaches

CRISPR/Cas9 technology is a revolutionary tool in the field of molecular biology that has opened up new possibilities for the treatment of genetic diseases. It is a genome-editing technology that uses a guide RNA to target specific DNA sequences and a Cas9 enzyme to cut and modify the DNA. The potential of CRISPR/Cas9 technology in treating genetic diseases, including NF1, has generated considerable excitement in the scientific community.

CRISPR/Cas9 technology offers the possibility of correcting the genetic mutations that cause NF1 by precisely targeting and repairing the defective genes. In preclinical studies, researchers have used CRISPR/Cas9 technology to correct mutations in the NF1 gene in vitro and in animal disease models. For example, a study published in 2018 demonstrated successful correction of the NF1 gene mutation in Schwann cells, the cells that produce the myelin sheath that surrounds and protects nerve fibres [44]. The researchers used CRISPR/Cas9 to insert a functional copy of the NF1 gene into the cells, resulting in increased neurofibromin protein levels and improved myelination.

Another study published in 2018 demonstrated the potential of CRISPR/Cas9 to treat pains caused by NF1 [45]. By editing the Nf1 gene using CRISPR/Cas9, CRMP2 has been identified as a potential target for treating pain associated with neurofibromatosis type 1. The research used of (S)-Lacosamide to reverse this pain.

Despite the promising results of these preclinical studies, there are still significant challenges to overcome before CRISPR/Cas9 technology can be used as a safe and effective therapy for NF1 patients. One of the major concerns is the potential for off-target effects, in which the guide RNA and Cas9 enzyme may inadvertently target and modify unintended genes, leading to

unintended consequences. Another concern is the potential for immune responses to the CRISPR/Cas9 components, which could limit the effectiveness and safety of the therapy.

Nonetheless, the potential of CRISPR/Cas9 technology in the treatment of NF1 is significant, and ongoing research is focused on addressing these challenges and developing safe and effective CRISPR/Cas9-based therapies for NF1 and other genetic diseases.

## 6.4 Exploring the Potential of miRNAs and IncRNAs as Therapeutic Targets

Recent studies have shown that non-coding RNA molecules, particularly microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), may play a crucial role in the development and progression of NF1, and could potentially serve as therapeutic targets for the disease.

MiRNAs are small RNA molecules that regulate gene expression by binding to messenger RNA (mRNA) and preventing their translation into proteins. Several studies have identified dysregulated miRNAs in NF1, including miR-155, miR-10b, and miR-150, among others. These miRNAs have been implicated in various cellular processes, including inflammation, cell proliferation, and differentiation, which are crucial for the development of neurofibromas. For instance, miR-155 has been shown to promote the growth of neurofibromas by regulating the expression of several genes involved in cell proliferation and survival, such as c-Myc and Bcl-2.

In addition to miRNAs, IncRNAs have also emerged as potential therapeutic targets for NF1. lncRNAs are longer RNA molecules that do not code for proteins but play crucial roles in gene regulation and cellular processes. Recent studies have identified several dysregulated IncRNAs in NF1, including HOTAIR, MALAT1, and ANRIL, among others [46]. These IncRNAs have been shown to regulate the expression of genes involved in various cellular processes, such as cell proliferation, apoptosis, and differentiation, which are crucial for the development and progression of neurofibromas.

The dysregulation of miRNAs and IncRNAs in NF1 suggests that these non-coding RNA molecules may play a critical role in the disease's pathogenesis and could serve as therapeutic targets. Several approaches have been proposed for targeting miRNAs and IncRNAs in
NF1, including the use of antisense oligonucleotides, small molecule inhibitors, and gene therapy. For instance, a study demonstrated the therapeutic potential of antisense oligonucleotides targeting miR-10b in a mouse model of NF1 [46,47]. The treatment led to a significant reduction in neurofibroma growth and improved neurological function in the mice.

## 6.5 Macrophage Therapy

The research focus in the neurofibroma tumor microenvironment has turned towards pNF-associated macrophages. In fact, macrophages are abundant in neurofibromas and MPNSTs, and their recruitment is stimulated by macrophage colony-stimulating factor (M-CSF). Studies have shown that inhibiting macrophages can slow down pNF progression, making them a potential therapeutic target for neurofibroma, glioma, and tumorous glial cells.

In the case of OPG, microglia have been investigated to determine their role in tumour growth. Results indicate that microglia promote the growth of OPG, making stromal cells such as microglia a potential target for treatment strategies [48]. Researchers discovered that the expression of numerous cytokines, chemokines, and their receptors is altered in microglia in the context of OPG, including the chemokine ligands CX3CL1 and Ccl5 [49]. However, the mechanism by which CCL5 promotes tumorigenic activity in the context of OPG remains to be demonstrated.

"Although macrophages are abundant in pNF and MPNSTs in both mice and humans, their absolute role in tumorigenesis in the NF1 context is not yet clear. Inhibiting the receptor for macrophage M-CSF successfully decreased macrophage density and promote tumor regression in mice, but only once pNFs had been established" [50]. "It is uncertain which cells and pathways within the tumor contribute to the reduced growth of pNFs in response to treatment, and a better understanding of the downstream effectors of ccl5 is needed. The Cxcl10-Cxcr3 chemokine axis appears to play a critical role in macrophage recruitment for neurofibroma formation, and pegylated IFN-α2b has been shown to slow down neurofibroma growth in some NF1 patients, warranting further investigation" [51].

## 7. CONCLUSION

Neurofibromatosis type 1 (NF1) affects one in every 3,000 individuals worldwide, leading to

benign tumor growth on nerves and various complications. Although there is no cure and current treatments are limited, recent breakthroughs in research offer hope for new therapeutic approaches.

Historically, surgery and symptom management were primary treatments. However, targeting molecular mechanisms like Ras and mTOR signaling has emerged as a promising strategy. MEK inhibitors, mTOR inhibitors, and immunotherapy have shown encouraging results in preclinical and clinical trials. Combining targeted therapies with personalized precision medicine, tailored to individual genetic alterations, holds potential for improved outcomes.

Further basic research is crucial for understanding NF1 pathogenesis, including the roles of microenvironmental cells like macrophages and microglia. Identifying new therapeutic targets through this research is essential for enhancing patient outcomes. The translation of basic research findings has driven significant progress in NF1 therapy. Ongoing efforts in this direction hold tremendous promise for enhancing the lives of individuals affected by NF1.

## CONSENT

It's not applicable.

## ETHICAL APPROVAL

It's not applicable.

## ACKNOWLEDGEMENTS

We thank Dr Jean-Philippe Brosseau for granting us permission to re-use the figures in his earlier published publication. We also thank Professor Volker Budach for granting us permission to re-use the figures in his earlier published paper.

## COMPETING INTERESTS

Authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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