From viral vectors to what’s next: The race to unlock targeted gene insertion

The first generation of cell and gene therapies was built on viral vectors—and for a time, they were revolutionary. AAV and lentivirus became foundational tools for delivering genetic material into human cells, enabling the approval of life-changing therapies like Zolgensma (for spinal muscular atrophy) and Kymriah (the first CAR-T therapy). But as the field matured, their limitations became harder to ignore. AAVs are size-capped (~4.7 kb), immunogenic, and complex to manufacture. Lentiviruses integrate semi-randomly, raising safety concerns—especially in pediatric populations. Both are expensive and poorly suited for broad, scalable gene insertion.

Over the past decade, the field took a leap forward with the rise of CRISPR and other editing tools. These systems offer precision and programmability—ideal for knocking out genes or making small corrections. The approval of Casgevy (Vertex/CRISPR Therapeutics) for sickle cell disease marked a major milestone. But CRISPR’s potential for inserting whole genes has yet to be realized at scale.

Why? Two key bottlenecks:

• DNA toxicity: Linear double-stranded DNA donor templates can trigger innate immune responses, especially in primary human cells.
• Nuclear delivery: Viruses evolved to solve this, but most non-viral systems don’t naturally enter the nucleus. AAV, for example, delivers single-stranded genomes that access the nucleus efficiently. That’s not true for most synthetic systems.

Because these problems remain unsolved, some companies have shifted strategy. Generation Bio, for instance, originally aimed to deliver non-viral DNA but ultimately pivoted to RNA-based therapies—which are easier to deliver and avoid DNA-related toxicity, but only operate in the cytoplasm and offer transient expression. These platforms may work for antibody or protein expression, but not for gene replacement or durable editing.

Now, the field is at an inflection point.

• Non-viral delivery technologies (e.g., LNPs) are catching up
• Editing tools like CRISPR, TALENs, and transposases are widely available
• But DNA payload design—and safe, efficient intracellular and nuclear delivery—remains the choke point.

The next wave of durable, scalable therapies—like site-specific CAR-Ts, in vivo gene correction for rare diseases, or programmable genomic insertions—requires innovation beyond the editing enzyme. The payload matters. DNA rises from raw material to therapeutic gene product.

Entering the 2nd decade of CGT – should we continue to believe in market growth?

We’re often asked: is Cell and Gene Therapy (CGT) just hype, or are we watching the birth of a lasting therapeutic category? To answer that, it helps to look back — at antibodies. Using sales data from EvaluatePharma, we can overlap the antibody market sales with CGT market sales and projections.

When monoclonal antibodies were first commercialized in the mid-1990s (with ReoPro in 1995), they too faced real skepticism. Yields were low, manufacturing was complex, and early applications were narrow. But as production capabilities improved — from ~0.1 mg/L to 5–10 g/L — the market scaled dramatically. By 2009, 15 years into its journey, the antibody market had grown to nearly $20B annually.

Today, CGT is showing a strikingly similar trajectory. Combined gene and cell therapy sales are projected to reach over $25B by 2030 — just 14 years after the launch of early drugs like Strimvelis (2016) and Luxturna (2017). The resemblance isn’t just in the sales curve — it’s in the evolution of manufacturing, clinical complexity, and regulatory confidence.

At Kano, we see this parallel as both validating and motivating. The CGT field is advancing rapidly, but like antibodies in the early 2000s, it’s being held back by manufacturability — especially when it comes to the DNA payloads required for more complex cell and gene edits. That’s where we come in.

• Antibody success was unlocked by improving yield and scalability — turning bespoke biologics into repeatable, high-throughput processes.
• We’ve taken cssDNA production from lab-scale (0.1 mg yield) to early scalable runs achieving up to 1 g/L crude material — and are continuing to optimize for downstream purity and throughput.
• By unlocking non-viral DNA design and manufacturing, we aim to enable next-gen gene insertion therapies that are currently just out of reach due to payload limitations.

CGT is entering its second decade. If the antibody playbook holds, the biggest breakthroughs are still ahead — and manufacturability will once again be the catalyst.

A closer look at CAR-T

Why CAR-T therapies should move from viral to non-viral

Most commercial CAR-T therapies today rely on lentiviral vectors to deliver a chimeric antigen receptor (CAR) transgene into T cells. While this approach has enabled groundbreaking treatments for hematologic malignancies, it has several limitations:

• Imprecise integration: Lentiviral vectors integrate semi-randomly into the genome, which can lead to variable expression levels, gene disruption, or oncogenic activation. In contrast, CRISPR-based approaches enable targeted integration of CAR constructs at safe harbor loci (like TRAC or CCR5), ensuring consistent expression and avoiding harmful insertional mutagenesis.
• Manufacturing complexity and cost: Producing GMP-grade lentivirus is expensive, time-consuming, and batch-variable. Non-viral CRISPR-based methods—especially those using electroporation of RNPs and DNA templates—simplify manufacturing and enable more rapid up to 9 months faster, scalable workflows.
• Control over editing: With CRISPR, it’s possible to insert into a CAR at a specific locus. This level of precision is not feasible with lentivirus or AAV alone.

Why multi-target CAR-T and dynamic functionality

Tumor heterogeneity and antigen escape are major causes of relapse in CAR-T-treated patients. Many cancers—especially solid tumors—downregulate or lose the target antigen under therapeutic pressure. Multi-target CAR-Ts and gene circuits address this by:

• Reducing antigen escape: Targeting two or more tumor-associated antigens simultaneously decreases the likelihood that the tumor can evade recognition.
• Improving durability: Dual or tri-specific CARs (or logic-gated CARs) can maintain pressure on heterogeneous tumors and improve long-term remission rates.
• Enhancing safety: Advanced designs like AND/NOT gates can restrict CAR-T activity to tumor-specific combinations, reducing off-tumor toxicity.

Together, non-viral CRISPR-based engineering and multi-target CAR design represent the next generation of cell therapies—more precise, adaptable, and better suited to tackle both safety and efficacy challenges across diverse cancers and scalable to other indication profiles.