Gene Therapies to Reduce Chronic Pain: Are we There Yet?

Keywords::

• chronic pain
• CRISPR
• gene therapy
• Na_V1.7
• pain management
• precision medicine

Pain is an evolved biological mechanism designed to let our brain know when a certain part of our body is damaged. Nociceptors are specialized neurons equipped with ion channels that detect and propagate the pain stimuli. However, pain can transition from acute to chronic, and lead to a diminished quality of life. It is estimated that 1.5 billion people worldwide suffer from chronic pain [1]. In the USA, approximately one in every five Americans suffers from chronic pain and about 20 million report severe pain that interferes with their daily activities [2]. These numbers are estimated to rise due to an increase in the elderly population, in diabetes patients and in cancer survival rates. In this pain crisis, the USA is spending more than US$560 billion annually due to pain care, which includes healthcare, addiction treatments and loss of productivity [3].

Despite the overwhelming amount of people suffering from pain, the number of efficacious options is limited. Treatments commonly available to those who suffer from pain are non-opioids, such as nonsteroidal anti-inflammatory drugs, glucocorticoids and paracetamol, although these are not efficacious in many forms of severe pain. Another potential treatment for chronic pain includes botulinum toxin injections, but unfortunately, botulinum toxin injections have only been US FDA approved for chronic migraines due to their lack of efficacy or potential side effects with general chronic pain [4]. Another potential treatment for chronic pain is epidural steroid injections, which consist of a mixture of corticosteroids and anesthetics into the epidural space to relieve pain for approximately 3 months. The most common treatment option for severe pain is opioids, which can be accompanied by a range of detrimental secondary effects and can be addictive [5]. In fact, the USA is currently undergoing an opioid epidemic. Roughly 21–29% of patients prescribed opioids misuse them, leading to 9% of patients developing a use disorder, and to about 128 people dying from opioid overdose each day in the USA [6,7]. We are in urgent need of new treatments for long-term pain management and gene therapies offer an attractive alternative approach based on the contribution of different genes to chronic pain.

Genetic factors account for 30–76% variance in pain response and can account for both pain threshold and susceptibility to chronic pain [8]. Understanding the mechanisms of rare chronic pain disorders in patients has facilitated the identification of subsets of genes involved with pain physiology and pathology [9]. Of particular interest are some members of the voltage-gated sodium channel family (Na_V). There are nine Na_V subtypes in vertebrates, of which Na_V1.7, Na_V1.8 and Na_V1.9 (SCN9A, SCN10A and SCN11A) have been implicated in pain transmission and enhanced hyperexcitability in nociceptive neurons. Characterization of mutations in these channels has confirmed a causative link of these channels to human pain disorders [10]. Patients with SCN9A loss-of-function mutations have insensitivity to pain without any other major effect apart from anosmia – deficit in the sense of smell [11]. In contrast, gain-of-function mutations in SCN9A have been correlated with erythromelalgia, a rare genetic disorder characterized by burning pain episodes [12]. Mutations in other members of the sodium channel family, such as Na_V1.8 and Na_V1.9, have also been correlated to pain [13]. Recent research into Na_V1.8 suggests its involvement with cold allodynia in neuropathic pain [14,15]. Additionally, patients with loss-of-function mutations in SCN11A show insensitivity to pain [16]. Another interesting report has correlated a loss of function in the FAAH gene (fatty acid amide hydrolase) with insensitivity to pain [17]. However, some neurological side effects were also reported. Due to the numerous correlations between genes and pain, gene therapies have emerged as a viable alternative to current existing pain therapies, with each gene therapy modality having its own advantages and disadvantages.

Traditionally, gene therapy has been used to replace a faulty gene; however, novel technologies have expanded our ability to target RNA and DNA without the need of replacing a gene. For instance, RNAi takes advantage of a conserved mechanism in eukaryotic cells shown to induce loss-of-function phenotypes [18]. Silencing is mediated by double-stranded siRNAs which lead to the degradation of their target mRNA and thus a reduction in the amount of protein encoded. Indeed, some manuscripts report that the knockdown of SCN9A with shRNA ameliorates pain in rodent models of burn injury and bone cancer [19,20]. However, siRNAs are easily degraded by RNases, can produce undesired off-target effects, and induce unspecific immune responses. Even though RNAi technology has been known for quite some time, only two RNAi drugs have been approved by the FDA [21].

Another therapeutic tool is antisense oligonucleotides (ASOs), which are short, synthetic, single-stranded oligodeoxynucleotides that can interfere with mRNA processing, leading to endonuclease-mediated protein silencing [22]. These ASOs often have off-target toxic effects and insufficient and inadequate target interactions. Although improvements have been recently made, cellular uptake and toxicity persists to be a major issue [22]. A recent study has utilized ASOs to target SCN9A and demonstrated that mechanical pain could be ameliorated with 30–80% SCN9A repression levels, however, no toxicity or specificity analysis was reported [23]. Another study showed significant reduction of tactile allodynia and thermal hyperalgesia in rats with chronic compression of the dorsal root ganglia after intrathecal administration of ASOs targeting T-subtype calcium channels [24]. Even though the FDA approved the first ASO therapy more than 20 years ago, as of now, only seven have been approved for rare diseases. The short half-life – compared with other gene therapies – and biodistribution issues, has hampered its broader use in chronic pain thus far.

As groundbreaking technologies emerge in the biological field, creative ideas to tackle health issues have surfaced. For instance, chemogenetics aims at redesigning channels or other macromolecules so that they recognize synthetically made small molecules rather than their endogenous counterparts. This approach would allow selective control of the excitability of neuronal subpopulations. Although promising, expressing these semisynthetic channels in sensory neurons of chronic pain patients may prove difficult, and side effects can come from the long-term (cumulative) administration of synthetic small molecules and from the new macromolecule itself. Recently, the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 has enabled scientists to target genes in a more specific and direct approach at the DNA level. However, a permanent reduction of the perception of pain is not desirable, so the use of a catalytically inactive ‘dead’ version of Cas9 enzyme (dCas9 also known as CRISPRi) with a guide-RNA and a repression domain – Krüppel-associated box (KRAB) – allows the binding of the target gene without permanent genome editing [25]. Thus, the CRISPRi-KRAB approach takes advantage of the dCas9 targeted specificity while ensuring no genomic mutations are made and decreases the transcription levels of the gene of interest.

CRISPRi-KRAB allows for greater specificity, increased therapeutic longevity and decreased application frequency than the other approaches aforementioned. Two studies have shown that the off-target effects of RNAi, as compared with CRISPRi, are far stronger and more prevalent than generally appreciated [26,27]. While RNAi and ASO therapies target the RNA molecules, reducing the later protein translation, dCas9 targets the DNA, modifying transcription levels. In theory, each cell would only need two CRISPRi-KRAB complexes – one for each allele – to repress the majority of target gene expression. Conversely, the RNA-targeting approaches may require higher number of molecules per cell due to RNA turnover and more frequent application to obtain a similar effect. Alternatively, a similarly promising approach is to use zinc finger proteins (ZF) fused to a KRAB repressor [25]. ZFs are among the most abundant proteins in eukaryotic genomes [28] and the Cys₂His₂ ZF DNA-binding domain is the most common domain in humans. In addition, the DNA recognition capacities of ZFs have shown similar results as CRISPRi to target Na_V1.7 [25]. The major advantage of ZFs is that they are endogenous to humans and thus would cause lower immune responses than dCas9, which is prokaryotic in origin. Conversely, the main drawback of ZFs is that they require deep protein bioengineering skills to design potent ZFs and modifications to increase specificity are laborious.

Delivery of the chemogenetic macromolecule, dCas9 or ZFs to patients in a clinical setting would require a delivery vector and adeno-associated virus (AAV) are the preferred vectors. Advantages of using AAV as a delivery vehicle is their innate ability to enter and deliver genetic information to host cells, their long-term transgene expression and their low immune response and toxicity. Thus, two AAV serotypes have already been approved by the FDA – AAV2 and AAV9 – and an ever-growing number of clinical trials are ongoing [29,30]. However, a major limiting factor is their restricted packaging capacity. Cas9 variants are rather large proteins, which would limit the additional regulatory elements – repressor domains, promoters, enhancers, among others – that can be inserted into the same transgene. In contrast, ZFs proteins are usually smaller than any Cas9 variant, which would allow flexibility in additional regulatory elements.

Although gene therapy-mediated treatments for severe pain are promising, various challenges lie ahead. The first is manufacturing costs. Manufacturing of components needed in gene therapies can be time consuming, difficult to produce in high yields, costly, and due to the increasing demand for gene therapy manufacturing, can take a long time to produce, which would make the therapy inaccessible for many patients. In 2019, the FDA approved a $2.1 million gene therapy for pediatric patients with spinal muscular atrophy [31], and many patients could have a hard time gaining access to these groundbreaking and sometimes life-saving treatments. Another challenge would be the immune responses against systemic delivery of AAV. To mitigate these effects the AAV vector could be administered into the spinal intrathecal space. Intrathecal injections would bypass the blood–brain barrier and target pain stimuli before it reaches the brain, decreasing the possibility of systemic immunogenicity, and reducing the dose necessary to achieve pain amelioration. Another bottleneck in gene therapies is the drug approval process. As with any precision medicine drug, approval for novel gene therapies is not as straightforward and properly guided as with traditional small molecules or antibodies.

Conclusion

Overall, due to the limited number of efficient medicines for chronic pain, there is an urgent need to expand the repertoire of treatments for chronic pain. Studies that have correlated the contribution of genes to the pain process and novel therapeutic approaches to relieve pain have paved the way for a new era of potential alternative treatments. CRISPRi-KRAB and ZF-KRAB show great promise because of their high specificity, ability to target genes at the DNA level and long-lasting effects. However, before these precision medicine therapies are accessible to the public, gene therapy manufacturing improvements, a better understanding of the immune responses and a clear regulatory pathway will be needed.

Financial & competing interests disclosure

All authors are employees of Navega Therapeutics. Patents related to this study have been submitted. This work was partially supported by NIH grants R43CA239940-01 and R43NS112088-01A1. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

All authors are employees of Navega Therapeutics. Patents related to this study have been submitted. This work was partially supported by NIH grants R43CA239940-01 and R43NS112088-01A1. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.