Beyond the Payload

Over the last decade, cell and gene therapies (CGTs) have shown the ability to modify disease-inducing biological pathways, restore missing cellular functions, and achieve long-term therapeutic benefit following a single administration. However, even with significant design and development advancements, efficient on-target delivery remains challenging. The ability to safely, efficiently, and repeatedly deliver the correct genetic cargo to the right cell is one of the most critical determinants of clinical success.

While payload design will remain a primary focus, the next era of innovation in CGT may ultimately be driven by the delivery vehicle itself. In many cases, therapeutic efficacy, safety, and manufacturability are directly influenced by delivery performance. As payload technologies continue to advance, delivery may emerge as the key factor separating scientific possibility from clinical reality.

As a follow-up to last month’s article, “For mRNA, Delivery Is the New Standard,” which explored the future of mRNA delivery, this article examines the landscape of CGT delivery technologies. Specifically, we examine the strengths, challenges, and future directions of several leading platforms that are poised to play key roles in the future of advanced therapeutics.

Adeno-associated viral vectors (AAVs), lentiviral vectors (LVVs), and lipid nanoparticles (LNPs) have emerged as leading delivery platforms, each offering unique advantages and limitations. While AAV has largely dominated in vivo gene therapy, LVV has emerged as a viable in vivo delivery approach, in addition to its predominant use for ex vivo cell engineering applications, such as CAR T therapy. LNPs gained global recognition through their deployment in mRNA vaccines during the COVID-19 pandemic and have become a leading non-viral delivery technology. Because each platform employs a fundamentally different approach for transporting genetic material, complementary roles exist within the CGT landscape. Nevertheless, future advances for each platform will likely depend less on the genetic payload and more on improving delivery efficiency, target specificity, and safety.

Understanding the various delivery approaches

AAV is a small, non-enveloped viral vector that is approximately 20–25 nm in diameter. Although the overall structure between serotypes is similar, they vary significantly in receptor and surface proteins, which impact tissue targeting and immune recognition. Encapsulated within the capsid is a single-stranded DNA (ssDNA) genome containing the gene of interest (GOI). AAV does have a relatively limited packing capacity of around 4.7 kb, restricting the size of transgenes that can be delivered. Following cellular uptake and nuclear entry, the AAV genomic DNA is converted to double-stranded DNA and expressed by host-cell machinery to produce therapeutic proteins.

LVV contains a lipid envelope that mediates cell entry and encloses an RNA genome along with the viral enzymes required for genomic integration. Compared with AAVs, LVVs possess a larger packaging capacity and can typically accommodate genetic payloads of approximately 8–10 kb, enabling delivery of larger or more complex transgene constructs. Following cellular entry, the RNA genome is reverse transcribed into DNA and transported to the nucleus, where it integrates into the host genome. This permanent integration supports long-term transgene expression, which is unique to LVV as compared to the other delivery systems.

LNPs are synthetic, non-viral delivery systems that are typically 50–150 nm in diameter. These particles contain a dense core of ionizable lipids, phospholipids, cholesterol, and PEG-lipids that encapsulate a genetic payload, such as mRNA or siRNA. LNPs offer the greatest payload flexibility among the three platforms and can accommodate large mRNA molecules, combinations of nucleic acids, and gene-editing cargoes that may exceed the packaging limitations of viral vectors. Unlike viral vectors, an LNP does not deliver its genomic payload to the nucleus. Following cellular uptake and endosomal escape, the released mRNA is translated directly in the cytoplasm to the therapeutic protein.

Target specificity

Target specificity is becoming an increasingly important consideration as CGT payloads grow more powerful and complex. Emerging technologies, such as CRISPR, base editing, and prime editing, introduce new challenges due to their size, complexity, and heightened safety risks. As a result, the future success of many advanced therapies may depend not only on what they deliver, but how precisely they can deliver the cargo.

For in vivo delivery, AAV offers the highest degree of target specificity and can effectively target many different cell types. Different serotypes naturally exhibit different tissue tropisms, and capsid engineering efforts have further enhanced the ability to selectively target specific tissues. AAV vectors are widely regarded as the most mature platform for targeted in vivo gene delivery. As regulatory expectations continue to emphasize safety, the ability to precisely direct therapeutic payloads may become even more valuable.

While LVVs generally contain moderate target specificity in vivo, they have demonstrated extraordinary success in ex vivo applications, in which target cells are isolated, genetically modified, and returned to the patient. The success of in vivo LVV therapy will likely depend on continued improvements in targeting technologies, including custom envelope engineering, which may require a shift away from the frequently used VSV-G glycoprotein.

When compared with AAV and LVV, LNP generally has the lowest intrinsic target specificity and the highest degree of off-target delivery. Following systemic administration, LNPs have displayed a tendency to accumulate in the liver, limiting their ability to selectively target other tissues. However, advances in lipid chemistry and targeting strategies have shown promise for improving tissue specificity.

Safety

Improved targeting and delivery efficiency can be directly linked to improved safety. In many advanced therapies, high doses are required to achieve sufficient therapeutic activity. However, these elevated doses increase off-target risks, toxicity, and unintended immune responses. Because of this, improved delivery efficiency can improve safety by lowering dose requirements.

AAV vectors are generally well tolerated and have become the leading platform for in vivo gene therapies. Pre-existing immunity against naturally occurring AAV serotypes, however, can reduce efficacy and complicate (re)dosing strategies. High dosing requirements have been linked with serious adverse events, including hepatotoxicity, thrombotic microangiopathy, cytokine-mediated toxicity, and sensory neuron toxicity.¹ These findings have increased regulatory focus on dose optimization, patient selection, and immune monitoring.

LVV is often preferred because its ability to integrate into the host genome can provide durable gene expression, even as treated cells divide and pass the therapeutic sequence to daughter cells. This long-term persistence can be particularly advantageous in therapies targeting proliferating cell populations. However, permanent integration also introduces safety concerns, including the risk of insertional mutagenesis, that do not apply in the same way to non-integrating CGT platforms. Modern self-inactivating LVVs have substantially reduced this concern, yet long-term monitoring remains important.²

LNPs avoid the genomic integration risks associated with viral vectors and provide only transient expression of their RNA cargo. Although LNPs contain no viral proteins, both the lipid components and the RNA payload can stimulate innate immune responses that contribute to vaccine efficacy and common side effects. LNPs also have relatively high off-target delivery rates, particularly to the liver, that carry additional safety risks. Although uncommon, other risks associated with LNPs are PEG-associated immunogenicity and lipid-associated toxicity.³  Some of these risks are amplified in applications requiring repeat dosing, which is increasingly common for RNA-based therapies.

Future directions

The future of CGT is expected to see continued advancements in the coming years. Some of the most transformative developments may come from improvements in delivery technology. These innovations may come in the form of enhanced target specificity and expanded delivery of sophisticated payloads, such as CRISPR, base editing, prime editing, and other emerging technologies. Successful progression will rely on the development of delivery systems capable of precisely transporting these payloads to the appropriate cells while minimizing safety risks.

For AAV, future innovation will likely focus on engineered capsids with improved transduction efficiency, enhanced tissue specificity, and reduced susceptibility to pre-existing immunity. These advancements could support lower dosing strategies, which are generally associated with increased safety and decreased manufacturing demands, while also expanding the range of treatable diseases. AAV is also being pursued as a delivery system for gene editing technologies, such as CRISPR. While the high specificity makes AAV a valuable option, the payload limitations introduce challenges. Researchers are working on new ways to overcome this hurdle, such as engineering variants that yield smaller machinery proteins or using dual-AAV systems that split large transgenes into separate vectors.

For LVVs, the future will likely involve continued dominance in ex vivo applications while further expanding into in vivo gene delivery. Achieving that transition will require new envelope designs and targeting approaches capable of delivering genetic payloads with greater precision. If custom-engineered envelopes can develop LVVs that preferentially target specific cells at a high rate, the large payload capacity of LVVs would make them prime candidates for delivering gene editing tools.

For LNPs, advances in lipid chemistry and surface engineering may unlock applications far beyond the liver. If LNPs can be designed to effectively achieve this extrahepatic delivery, a plethora of new uses would potentially be unlocked. Because of their flexibility, scalability, and compatibility with gene-editing systems, LNPs are particularly well positioned to support future generations of RNA and genome-editing therapies. Lastly, innovations can be made to enhance LNP efficiency by improving the endosomal escape issue that plagues these delivery systems.

AAV, LVV, and LNP delivery platforms are all expected to remain central to the future of CGT. AAV remains the leading platform for targeted in vivo delivery, LVV offers unique advantages through its payload capacity and durable expression, and LNPs provide unmatched flexibility and scalability. Rather than a single dominant technology emerging, future success will depend on selecting the most appropriate platform for specific therapeutic objectives. As delivery systems continue to mature, ensuring consistent product quality and analytical characterization will become increasingly important for their successful development and adoption.

Whether utilizing AAV vectors, LVVs, or LNPs, future innovations will require equally sophisticated analytical tools to evaluate quality attributes. To support emerging technologies and new modalities, USP actively engages with stakeholders from across industry, academia, and regulatory communities to identify key challenges and areas where standards can best support adoption and improve consistency. Reference standards can help laboratories develop methods and monitor performance. USP has already developed a portfolio of AAV reference materials that support a wide range of analytical workflows and has recently expanded into LVV reference materials, with additional LVV- and LNP-related standards under development. As the field continues to advance, these standards can help ensure consistent and accurate characterization needed to support development and improve confidence in product quality.

Conclusion

As gene therapies become more sophisticated, including the incorporation of gene- and base-editing technologies, efficient and precise delivery will remain a key determinant of clinical success. Continued advances in targeting, payload delivery, manufacturability, and safety are expected to expand the capabilities of AAV, LVV, and LNP platforms. Rather than competing technologies, these delivery systems are likely to serve complementary roles, each offering distinct advantages for specific therapeutic applications. Ultimately, the next generation of transformative medicines may be defined not only by the genetic payloads they carry, but by the ability to safely, efficiently, and precisely deliver those payloads to the right cells at the right dose.

Anthony Blaszcyk, PhD, is principal scientist in the Global Biologics Department at USP.

References

1. Ertl HCJ. Mitigating Serious Adverse Events in Gene Therapy with AAV Vectors: Vector Dose and Immunosuppression. Drugs. 2023;83(4):287-298. doi:10.1007/s40265-023-01836-1.
2. Schlimgen R, Howard J, Wooley D, et al. Risks Associated with Lentiviral Vector Exposures and Prevention Strategies. J Occup Environ Med. 2016;58(12):1159-1166. doi:10.1097/JOM.0000000000000879.
3. Kim S, Park C, Lee Y, et al. Inflammatory and immune-mediated toxicity of lipid nanoparticles. Biomed Pharmacother. 2026;199:119385. doi:10.1016/j.biopha.2026.119385.

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