Breaking Barriers: PackGene Facilitates Gene Therapies for OTOF Hearing Loss and SPG56

Rare diseases, also known as orphan diseases, are medical conditions that affect a small percentage of the population. The classification of a disease as “rare” can vary by region. In the United States, a disease is classified as rare if it affects fewer than 200,000 individuals. In the European Union, the threshold is set at fewer than 1 in 2,000 people. Other countries have similar definitions, tailored to their respective healthcare systems.

Despite their individual rarity, collectively rare diseases represent a significant public health concern due to the diversity and complexity of the conditions involved. They can be genetic, infectious, degenerative, or idiopathic in nature, and often have a substantial impact on patients’ lives, leading to chronic illness, disability, and in some cases, premature death. An estimated 3–6% of the global population—hundreds of millions of people—live with one of these conditions. In the United States alone, approximately 30 million individuals are affected by one of the roughly 7,000 identified rare diseases, highlighting the pervasive nature of these often-underrecognized disorders. These conditions, spanning genetic anomalies to complex syndromes, impose a significant burden on patients, families, and caregivers, who face challenges such as scarce treatment options, diagnostic uncertainties, and considerable emotional and financial strain. The scope of this issue continues to expand, with around 200 new rare diseases identified annually.

The genetic roots of many rare diseases provide a lens through which to examine distinct yet complementary conditions like Hereditary Spastic Paraplegia Type 56 (SPG56) and genetic hearing loss, both of which illustrate how inherited mutations can profoundly affect children, albeit through divergent pathways—one impacting motor function and the other sensory capabilities. SPG56, an ultra-rare autosomal recessive disorder caused by mutations in the CYP2U1 gene on chromosome 4q25, affects fewer than 1 in 1,000,000 individuals worldwide, with fewer than 30 cases reported since its discovery in 2012. It typically emerges in early childhood, marked by progressive lower-limb spasticity that may extend to the upper limbs, sometimes accompanied by cognitive impairment or dystonia. By contrast, genetic hearing loss represents a far more prevalent genetic challenge, contributing to hearing impairment in 50% to 60% of the 34 million children affected globally out of the 466 million total cases of hearing loss, according to the World Health Organization. In developed countries, congenital hearing loss strikes 1 to 2 per 1,000 newborns. In the U.S., this impacted over 6,000 infants in 2022. Together, these examples underscore the diverse manifestations of genetic disorders and the pressing need for targeted interventions, public health strategies, and awareness to address both rare and widespread conditions shaping human health.

Gene therapy has emerged as a groundbreaking approach for treating rare genetic diseases, offering the potential for long-term or even curative outcomes. Among the various delivery systems, adeno-associated virus (AAV) vectors are widely used due to their ability to efficiently target cells with relatively low immunogenicity (Figure 1).

However, despite its promise, the development of gene therapies for rare diseases faces significant hurdles. Here are the key challenges:

1. Scientific Complexity and the Pervasive Unknowns

The elusive nature of rare diseases stems from a tangled web of scientific complexities and vast, uncharted territories. Diagnostic hurdles abound, as varied and overlapping symptoms obscure clear identification, exacerbated by the sheer volume of these conditions and the difficulty in securing research samples. The intricate genetic underpinnings, with numerous mutations contributing to each disease, further confound understanding. Limited research, hampered by small patient pools and funding scarcity, also slows progress.

a. Hearing Loss

Hearing loss is the most common sensory disorder in humans, with the majority of congenital cases linked to genetic mutations. Over 150 genes and 6,000 variants have been associated with hereditary hearing loss, highlighting its extreme genetic heterogeneity. This complexity stems from the intricate anatomy and physiology of the auditory system, particularly the inner ear, which comprises sensory hair cells, supporting cells, spiral ganglion neurons, and the stria vascularis. These components work in concert to convert sound waves into electrical signals for the brain. Genetic mutations can disrupt any part of this process—from hair cell development and function to synaptic transmission—resulting in diverse phenotypes, including conductive, sensorineural, syndromic, or nonsyndromic hearing loss. The key challenges in addressing genetic hearing loss include:

• • Diversity of Genetic Mutations: Genetic hearing loss can be monogenic (caused by a single gene) or polygenic (involving multiple genes and environmental factors). Monogenic forms, such as those caused by mutations in the otoferlin gene (OTOF), are more straightforward targets for gene therapy, as they involve replacing or repairing a single defective gene. Polygenic forms, however, pose a greater challenge due to the need to address multiple genetic and environmental interactions.
  • Inner Ear Accessibility: The cochlea, encased in the temporal bone, is difficult to access without risking damage to its delicate structures. Delivery of gene therapy vectors—typically AAVs—requires precise surgical techniques, such as injection through the round window membrane or cochleostomy, which must balance efficacy with safety.
  • Timing of Intervention: Many genetic defects cause irreversible damage to hair cells or neural structures before or shortly after birth. For example, in congenital deafness, hair cell degeneration may occur prenatally, necessitating intervention during fetal development—a prospect fraught with ethical and technical hurdles.

b. SPG56

SPG56, caused by mutations in the CYP2U1 gene, is a rare form of hereditary spastic paraplegia (HSP). HSPs are a group of inherited neurological disorders characterized by progressive spasticity and weakness of the lower limbs, resulting from degeneration of the corticospinal tract neurons. Among more than 80 identified genetic subtypes, SPG56 stands out as an ultra-rare variant, with fewer than 30 families reported worldwide.

• • Molecular Function of CYP2U1: The CYP2U1 gene encodes a cytochrome P450 enzyme that metabolizes long-chain fatty acids, such as arachidonic acid, into bioactive lipids critical for cellular signaling and mitochondrial function. Mutations typically result in a loss of function, disrupting neuronal homeostasis—particularly in corticospinal tract neurons responsible for motor control. However, the enzyme’s dual localization (endoplasmic reticulum and mitochondria) and its broad substrate specificity mean its precise role in neuronal health is multifaceted and not fully mapped, complicating therapeutic restoration.
  • Genetic Heterogeneity: Over 25 distinct CYP2U1 mutations (missense, nonsense, frameshift) have been identified, often leading to truncated or inactive proteins. This diversity requires a tailored approach—either a universal strategy to replace the gene or mutation-specific fixes, both of which add layers of complexity to therapy design.
  • Phenotypic Variability: SPG56 ranges from pure spasticity to complicated forms with cognitive decline, dystonia, and white matter abnormalities. This heterogeneity suggests that simply restoring CYP2U1 function might not address all symptoms if secondary pathways (e.g., mitochondrial dysfunction or lipid signaling cascades) are irreversibly damaged by the time therapy is administered.
  • Overlap with Other Disorders: Mutations in CYP2U1 have been linked not only to SPG56 but also to pseudoxanthoma elasticum and other neurodegenerative conditions, reflecting shared pathways (e.g., mitochondrial dysfunction or lipid signaling). This overlap complicates differential diagnosis and suggests CYP2U1 may have broader physiological roles than currently understood.

2. Challenges in Clinical Development

Translating preclinical success into human therapies requires robust clinical trials but recruiting patients and designing effective trials for genetic rare diseases present significant challenges.

a. Patient Recruitment

Patient recruitment for clinical trials targeting rare genetic conditions like SPG56 and OTOF-related deafness presents a formidable challenge due to their low prevalence, diagnostic barriers, geographic dispersion, and ethical complexities. SPG56 has a prevalence of less than 1 in 1,000,000, while OTOF-related deafness, accounting for just 1-8% of congenital hearing loss cases, is similarly scarce at rates as low as 1 in 500,000. Identifying and enrolling sufficient participants is a significant challenge, as the limited number of documented cases—fewer than 30 families for SPG56 globally—makes it difficult to assemble viable cohorts. Diagnostic hurdles further complicate recruitment: genetic testing, essential for molecular confirmation, is not universally accessible, and conditions like OTOF deafness may evade early detection, delaying diagnosis until symptoms are severe and trial eligibility is harder to establish. The CHORD trial, a Phase 1/2 clinical study conducted by Regeneron Pharmaceuticals and Decibel Therapeutics, is investigating the gene therapy DB-OTO for children with hearing loss caused by mutations in the OTOF gene. This multi-center trial, spanning the UK, US, and Spain, reflects the global distribution of patients with this condition; such multi-center trials escalate costs and coordination efforts. Ethical considerations add another layer of difficulty, particularly for pediatric populations common in these congenital disorders; parents must navigate the uncertainties of experimental interventions, such as gene therapy requiring invasive procedures, against established alternatives like cochlear implants for deafness or supportive care for SPG56, balancing potential benefits with inherent risks. Together, these factors make patient recruitment a critical bottleneck in advancing treatments for these rare diseases.

b. Trial Design and Endpoint Selection

Designing clinical trials for rare genetic conditions like SPG56 and OTOF-related deafness is inherently complex due to the diverse genetic and phenotypic profiles these disorders present, posing significant challenges to achieving consistent, meaningful outcomes. For example, a gene therapy tailored to specific OTOF mutations might effectively address that subtype of deafness but fail to benefit cases of polygenic hearing loss. This diversity forces researchers to either design separate trials for each genetic variant—an approach constrained by limited patient numbers—or adopt a basket trial framework that groups similar conditions together, though this sacrifices statistical power due to small, heterogeneous cohorts. Adding to the complexity, trials must optimize critical variables such as vector dose (e.g., low versus high) and delivery method (unilateral versus bilateral). The CHORD trial exemplifies this cautious approach, beginning with a low dose in one ear and escalating only after safety is confirmed, a strategy that extends timelines and increases resource demands.

Further complicating trial design is the need to understand the natural history of these conditions and select endpoints that reliably measure gene therapy efficacy—both of which are fraught with obstacles. Comprehensive longitudinal data is often scarce. For SPG56, the lack of detailed studies on onset, progression, and variability leaves significant knowledge gaps. Even for OTOF-related deafness, which is relatively better characterized, natural history records remain incomplete, particularly for patients diagnosed later in life. Selecting appropriate endpoints presents its own set of challenges. Objective measures like auditory brainstem response (ABR) and pure-tone audiometry, as utilized in trials like DB-OTO, can track neural improvements but may overlook subtle functional benefits. When it comes to SPG56, disease heterogeneity, slow progression, the extremely small patient pool, and the absence of baseline data or biomarkers would pose more significant challenges to endpoint selection in a clinical trial.

3. The AAV Manufacturing Hurdles

AAV gene therapy represents the primary therapeutic strategy for genetic diseases such as OTOF hearing loss and SPG56. However, the translation of AAV’s therapeutic potential into widespread clinical application is significantly hindered by manufacturing challenges, particularly those related to cost and scalability. These challenges arise from the intricate biological characteristics of AAV, the labor-intensive nature of its production processes, and the necessity for high-quality, high-titer vectors to meet clinical demands. Below are the main cost and scalability challenges:

a. High Input Costs for Raw Materials

Producing recombinant AAV (rAAV) typically involves transient transfection of mammalian cells (e.g., HEK293) with multiple plasmids: one encoding the therapeutic transgene, another providing AAV replication and capsid (rep/cap) genes, and a third supplying helper virus genes (e.g., from adenovirus). The reliance on high-quality, clinical-grade plasmid DNA is a major cost driver. Producing sufficient quantities of Good Manufacturing Practice (GMP)-compliant plasmids requires specialized facilities and rigorous quality control, inflating expenses. Additional raw materials, such as transfection reagents (e.g., polyethyleneimine or proprietary lipids), chemically defined media, and single-use bioreactors, further escalate costs. For therapies targeting larger patient populations, these inputs must be scaled proportionally, amplifying the financial burden.

b. Low Production Yields

Traditional AAV production methods are inherently inefficient. Successfully delivering three plasmids into a single cell is a stochastic process, often resulting in low yields of full, functional AAV capsids (typically 10^4–10^5 viral genomes per cell). A significant portion of capsids produced are empty or partially filled, lacking the therapeutic transgene, which reduces the effective yield and increases the cost per usable dose. Purification losses compound this issue. Separating full capsids from empty ones and removing contaminants (e.g., host cell proteins, DNA, and lipids) often relies on labor-intensive methods like density ultracentrifugation or chromatography, both of which can sacrifice yield for purity, driving up costs.

c. Downstream Processing and Scaling Constraints

Purification methods like density ultracentrifugation, while effective for small-scale research, are not scalable due to their low throughput and equipment demands. Alternatives like affinity or ion-exchange chromatography are being adopted, but they require optimization for each AAV serotype and can struggle to handle the increased volumes needed for commercial production. The trade-off between yield and purity persists, complicating efforts to scale without compromising quality. Removing impurities and separating full from empty capsids at scale remains technically challenging. Scaling AAV production introduces variability in critical parameters—cell growth, transfection efficiency, and vector stability—which can affect titer, potency, and purity. Suspension cultures in bioreactors (e.g., 200–2000 L) demand precise control of pH, dissolved oxygen, and shear stress, all of which can impact AAV yield if not optimized. Scalability limitations further slow clinical trials and commercialization, particularly for high-dose indications like systemic delivery for muscular dystrophy.

d. Supply Chain and Infrastructure Barriers

The global demand for AAV vectors has strained supply chains for critical components like plasmids, transfection reagents, and single-use bioreactor systems. Long lead times and shortages, as seen during the COVID-19 pandemic, can delay production schedules. Scaling to meet commercial needs requires not just process innovation but also expanded manufacturing capacity—new facilities or retrofitted plants—which entails significant capital investment and time.

4. PackGene Solutions for Cost and Scalability Issues

AAV manufacturing directly impacts the accessibility and commercialization of gene therapies. For ultra-rare diseases, where patient populations are small, high-cost small-scale production may be justifiable, but the exceptionally limited market size makes commercialization extremely challenging. Without significant financial incentives, pharmaceutical companies may be reluctant to invest in developing therapies for these conditions. Expanding gene therapy to more common diseases requires substantial cost reductions, necessitating breakthroughs in yield optimization, process efficiency, and standardization. Despite these challenges, our innovations in AAV gene therapy development aim to improve cost-effectiveness and scalability.

a. PackGene’s π-Alpha 293 AAV High-yield Platform

The AAV production platform significantly enhances AAV yields by utilizing a proprietary cell line that we recently developed. This system also includes a proprietary RC plasmid, designed to increase production 3-8x across various serotypes. Further optimization, guided by Quality by Design (QbD) principles in both upstream and downstream processes, achieves up to a 10-fold increase. This translates to a single-batch production output of 1E+17vg, a scale that readily meets the demanding requirements of clinical trials and commercial manufacturing, thus addressing key bottlenecks in AAV gene therapy development. The platform also reduces key impurity and enhances full/empty capsid ratio to improve biosafety and activity. Figure 2 shows that PackGene’s novel RC plasmid significantly increases the AAV production yield and provides high full/empty capsid ratio.

b. PackGene’s π-Omega Plasmid DNA High-yield Platform

This proprietary platform incorporates a sophisticated approach to plasmid backbone modification, resulting in a remarkable increase in plasmid yield—more than threefold. This enhancement directly translates to greater efficiency in downstream processes, as the increased availability of plasmid DNA significantly reduces production bottlenecks. Furthermore, the platform’s scalability is impressive, supporting production volumes of up to 200 liters. This large-scale capacity, coupled with a single-batch production output of up to 100 grams, positions the platform as a powerful tool for applications requiring substantial quantities of high-quality plasmid DNA, such as gene therapy manufacturing and large-scale research projects.

c. PackGene’s π-Icosa AAV Capsid Engineering Platform

This platform is designed to engineer and screen AAV capsid variants with enhanced properties—such as improved tissue specificity, higher infectivity, and reduced off-target effects—for gene therapy applications. It aims to address key limitations of natural AAV serotypes, which often lack the precision or efficiency needed for clinical success in diverse diseases. The platform integrates rational design, directed evolution, and high-throughput screening to create tailored AAV vectors, positioning it as a tool to optimize gene delivery for both research and therapeutic development. Utilizing this platform, we identified an AAV-PG008 variant exhibiting improved central nervous system target expression (Figure 3).

We are making significant strides in addressing rare diseases through its gene therapy manufacturing expertise and strategic collaborations. We are committed to delivering cost-effective manufacturing solutions for therapeutic materials, including AAV, mRNA, plasmid, and lentivirus. For OTOF-related hearing loss, PackGene’s provision of critical virus samples for RRG-OTOF, an in vivo gene therapy, was instrumental in securing FDA Orphan Drug Designation. This dual-vector approach aims to restore auditory function by delivering the otoferlin gene to cochlear hair cells. In the case of SPG56, an ultra-rare neurological disorder, PackGene is partnering with Genetic Cures for Kids and Weill Cornell Medicine to develop a clinical-grade gene therapy, pledging to subsidize costs and expedite development.

6. Conclusion:

At PackGene, we are committed to making gene therapies more accessible and affordable, particularly for ultra-rare conditions often overlooked by the pharmaceutical industry. By providing manufacturing solutions for plasmid DNA, and viral vectors, we are accelerating the development of tailored treatments for diseases affecting small patient populations. Our efforts aim to empower gene therapy developers, healthcare professionals, patient advocate groups, rare disease foundations, and all stakeholders in the fight against rare diseases.  Our work demonstrates that with scientific excellence and compassion, even the rarest conditions can benefit from cutting-edge gene therapies. We aim to create a replicable framework for addressing thousands of other genetic disorders currently without cures.

References
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17. ClinicalTrials.gov Identifier: NCT05788536. A Phase 1/2 Study of the Safety and Efficacy of OTOF Gene Therapy (CHORD Trial).
Latest DB-OTO Results Demonstrate Clinically Meaningful Hearing Improvements in Nearly All Children with Profound Genetic Hearing Loss in CHORD Trial. 2025 Feb. https://investor.regeneron.com/news-releases/news-release-details/latest-db-oto-results-demonstrate-clinically-meaningful-hearing

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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