By Elizabeth Figueroa, Ph.D., DHC Senior Consultant
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ABSTRACT
In vivo gene therapies have delivered astonishing results where few or no treatments have existed previously. Despite their curative potential, the field of gene therapy has not seen widespread adoption or effective commercialization. What are the challenges faced by in vivo gene therapies, and do non-viral vectors bridge these gaps?
In this white paper, we will address these questions by comparing adeno-associated virus (AAV) viral vectors (as representatives of current widespread in vivo viral technology) and lipid nanoparticle (LNP) non-viral vectors (as representatives of emerging non-viral technologies) at different phases of the product lifecycle. The emergence of non-viral vectors represents an opportunity to propel in vivo gene therapy further into wider commercial adoption and expand its impact globally. LNPs are poised to become the first commercially approved nonviral vectors for in vivo transgene delivery and genome editing approaches. While LNPs may face greater preclinical development hurdles than AAV, they are expected to have more streamlined clinical development and commercialization pathways, which will allow them to extend the scope of clinical indications accessible for treatment by in vivo gene therapies.
INTRODUCTION
Genetic diseases are caused by pathogenic genetic variants including point mutations, deletions, and duplications. In vivo gene therapies are a novel class of advanced therapeutic medicinal products that seek to address diseases through their underlying genetic cause. These therapies can deliver a transgene for therapeutic protein production, oligonucleotides for gene silencing, or a genome editor to permanently alter the genome. Transgene delivery approaches can increase production of a therapeutic or wild type protein or convey a new gain of function (GOF). Genome editing approaches can knockout or knock-in genes at specific sites in the genome, resulting in permanent gene silencing (e.g. indel-mediated gene knockout), gene correction (e.g. base editing), or gain of function (e.g. targeted whole gene insertion for production of a new gene).
[Watch Unbridled Excellence’s expert webinar #10 for more details on this topic, courtesy of Drs. Figueroa and Fuentes.]
Adeno-associated virus (AAV) based in vivo gene therapies for transient gene delivery dominate the current commercial cell and gene therapy (CGT) landscape in the United States. Despite over a decade of regulatory success, access to these therapies is constrained by sky high costs: up to $3.5MM per patient. Key drivers of high cost of goods sold (COGS) include biologically-derived raw materials, cold chain considerations for distribution, and complex manufacturing processes and analytics.
To fully realize the therapeutic potential of scientific advancements made in the identification of genetic targets and the development of genome editing tools, it will be critical to leverage cost effective, efficient, and safe delivery vectors. Whereas transgene delivery approaches have traditionally leveraged viral vectors such as AAV, the advent of genome editors has posed challenges for viral vectors due to the increased cargo size requirements and multiple delivery payloads associated with genome editing technology. Interest in non-viral vectors such as LNPs has risen due to their large cargo capacity, low immunogenicity, and low COGS. Some groups have even leveraged a combination of AAV and LNP approaches in the same drug product to facilitate delivery of DNA template and gene editors, respectively.
AAV and LNP vectors face very different challenges throughout the product development lifecycle which influence their commercialization potential. These challenges can be viewed through the lenses of preclinical development, clinical development, and commercialization phases.
Tables 1-3 provide a summary of the benefits and challenges associated with AAV viral vector and LNP non-viral vector approaches for in vivo gene therapies. The most important takeaway from such an assessment is that there is not a one-size-fits-all vector solution. While LNPs may face significant preclinical development hurdles, they are expected to demonstrate advantages in later stages of the product development lifecycle that far outweigh early-stage developmental challenges. Ultimately, LNPs have a rising competitive edge that may enable non-viral vectors to take a portion of the viral market share due to their low COGS, improved safety, and ease of manufacturing.
PRECLINICAL DEVELOPMENT
Looking more closely at preclinical development, LNPs are attractive for their versatile and unrestricted cargo capacity. Whereas the in vivo gene therapy gold standard of AAV can deliver DNA just under 5 kb, LNPs can encapsulate a variety of cargo including mRNA, DNA, siRNA, and proteins, even in combination, with as yet unrestricted cargo capacity. Additionally, LNPs’ transient expression profile, which for some transgene delivery applications may be less desirable, is considered a boon for genome editing from a safety perspective. For this reason, LNPs are potentially more well suited for genome editing applications which have larger cargo (looking at you, prime editors and gene writers) to achieve targeted, safe, and precise editing of the genome.
However, initial formulation development of LNP vectors is frequently challenged by a lack of model and structure-function relationship. To add to this challenge, mRNA-LNP formulation development is cargo- and target-dependent, necessitating screening of different lipid and mRNA ratios to optimize critical quality attributes (CQAs); even an off-the-shelf LNP formulation will require extensive development and optimization. Often, high throughput approaches are leveraged utilizing microfluidics, benchtop processes, or—more recently—barcoding approaches to increase the speed with which candidate LNP formulations can be identified. Then, several candidate formulations may still need to be screened across various nonclinical disease-relevant models to assess biodistribution and safety. If a novel lipid is leveraged in the LNP formulation, additional safety testing will be required to characterize the safety profile of the novel excipient. To add to this, biodistribution in some nonclinical models may not be predictive of biodistribution in humans.
The LNP supply chain is also complex, driven by the use of non-commodified reagents with non-trivial manufacturing process development. GMP lipid synthesis can require different synthesis pathways due to availability of precursor reagents, high levels of solvents and heat, and process residuals. Furthermore, purification can be challenging and biodegradability can impact manufacturing yields. On the bright side, though, LNP manufacturing is relatively straightforward when compared to any other CGT manufacturing process. LNPs are typically manufactured by self-assembly of lipids in an organic solvent with nucleic acid or protein cargo in an aqueous solvent, followed by purification, concentration, and filtration. In contrast, AAV production requires upstream manufacturing via a cell line that is transfected to produce AAV, followed by cell lysis, and downstream manufacturing involving purification, concentration, formulation, and fill finish. The amount of process variability and controls required for AAV production are significantly more extensive than LNP production, driven by biological process residuals and safety concerns, resulting in more process challenges and higher COGS.
CLINICAL DEVELOPMENT
From a clinical point of view, safety and targeting are the hot topics. Arguably the most critical challenge limiting clinical adoption of LNPs for in vivo gene therapy is improved biodistribution via systemic delivery. LNPs do not possess innate tropism, are subject to non-specific serum protein interactions, and are consequently susceptible to uptake by the reticuloendothelial system. These factors all amount to preferential accumulation to the liver, and as a result clinical in vivo gene therapy LNP applications thus far have been limited to targeting the liver, lungs, or blood. However, in vivo non-viral gene therapies in preclinical development expand that list to include ocular, lung, CNS, solid tumor and cardiovascular targets. This is often accomplished by conjugating the LNPs with antibodies, peptides, and other ligands that target tissue-specific antigens, as well as innovating upon novel lipid chemistry. Still, significant development is needed to achieve in vivo targeted delivery to other organs, necessitating innovation in LNP design to enhance trafficking across biological barriers.
However, LNPs possess an excellent safety profile, especially in relation to their viral counterparts. No need to assess potential for persistent expression or insertional mutagenesis, no concerns with pre-existing neutralizing antibodies, and the ability to re-dose LNPs all contribute to their appeal from a clinical safety perspective, as well as having a favorable impact on development costs and complexity.
COMMERCIAL PHASE
When considering their likeliness for broad adoption from a commercialization perspective, LNPs edge out AAV from a cold chain storage and distribution angle. The majority of AAV drug products must be stored at -60℃, whereas LNPs have been successfully lyophilized which can significantly reduce cold chain supply burden. The impact of storage at higher temperatures is increased access to patients beyond major metropolitan medical centers in western nations, which could allow for true globalized patient access and improved patient equity. Not only would this increase the profound clinical impact of in vivo gene therapies, especially for rare disease applications, but it would also result in a larger addressable patient population which allows developers to achieve more favorable return on investment (ROI) and manufacturing economies of scale.
And though the LNP intellectual property (IP) landscape is admittedly complex and presents a potential barrier to entry for developers – with mRNA, lipids, and the LNP formulations being readily subject to patents – the flip side is that development of a novel formulation provides an opportunity for developers to generate novel IP, with which they may pursue collaborations or licensing agreements to generate additional revenue streams. Alternatively, developers may opt to license a patented formulation to accelerate their development timelines, or if they do not have subject matter experts in house to pursue formulation development.
From a regulatory perspective, there is some uncertainty with respect to the designation of the lipids as an active ingredient, excipient, or drug substance in the LNP DP. While some may view lipids as inactive ingredients that stabilize the cargo, and for which a novel excipient classification would be appropriate, still others view the lipids as essential to the function of the LNP DP, therefore justifying their classification as an active ingredient or drug substance. The regulatory expectations moving forward should be monitored, as they will impact the degree of control and testing required for these non-commodified reagents.
CONCLUSION
Viral and non-viral approaches to in vivo gene therapy face unique product development challenges, including process development, supply chain, safety, manufacturing, and COGS. Non-viral platforms are safer and less costly to manufacture compared to viral vector platforms, whereas viral vectors benefit from their tissue tropism and delivery efficiency. AAV represents the gold standard of in vivo gene therapy vectors, but LNPs are poised to become the first commercially approved nonviral in vivo gene therapy vector. The low cargo capacity of AAV prevents delivery of larger cargo which may hinder its continued dominance, especially in the context of cutting-edge gene therapy approaches such as prime editors and whole gene insertion. It is anticipated that the preclinical hurdles unique to LNP product development will diminish as other LNP products are approved for extra-hepatic targets, establishing the clinical proof of concept that indeed preferential uptake beyond the liver is feasible. Widespread adoption and commercial success of in vivo gene therapies will be contingent on the ability to bring to the market safe, cost-effective, and efficacious drugs. Despite its current challenges, LNPs are poised to meet this requirement more effectively than AAV.
* For the purposes of this paper:
Nonclinical development is defined as characterization of product efficacy and safety using relevant in vitro and in vivo model systems, while preclinical development is defined as all development activities leading up to clinical manufacturing, including nonclinical development and manufacturing process development.
Many thanks to beta-reviewers Brent Morse, M.S., Christina Fuentes, Ph.D., and Anne Lamontagne, M.S.
