Spotlight Therapeutics: Transforming In Vivo Gene Editing

Gene editing holds the promise of treating genetic disorders at their root cause, but delivering these therapies directly into living organisms remains a challenge. Traditional approaches often rely on ex vivo modifications, where cells are edited outside the body and then reintroduced. In vivo gene editing—modifying genes directly within tissues—offers a more precise and potentially transformative solution.

Spotlight Therapeutics is advancing strategies to enhance the precision and efficiency of in vivo gene editing. By focusing on targeted delivery and cell-specific recognition, they aim to overcome existing barriers and expand the therapeutic potential of genome-editing technologies.

In Vivo Gene Editing Tools

Advancements in gene editing have led to sophisticated tools capable of modifying DNA sequences within living organisms. Unlike ex vivo approaches, which require cell extraction and reinfusion, in vivo gene editing necessitates precise molecular machinery that can navigate complex biological environments to reach target cells. The most widely used systems are CRISPR-Cas nucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), each offering distinct advantages in specificity, efficiency, and versatility.

CRISPR-Cas systems, particularly CRISPR-Cas9 and CRISPR-Cas12, have transformed genome editing due to their programmability and ease of use. These nucleases use a guide RNA to direct the Cas enzyme to a specific genomic locus, where it induces a double-strand break. The cell’s repair mechanisms—non-homologous end joining (NHEJ) or homology-directed repair (HDR)—then modify the DNA sequence. While NHEJ often results in small insertions or deletions that can disrupt gene function, HDR enables precise corrections when a repair template is provided. Recent innovations, such as base editing and prime editing, refine CRISPR-based tools by allowing single-nucleotide modifications without double-strand breaks, reducing the risk of unintended mutations.

ZFNs and TALENs, though less commonly used today, remain valuable for applications requiring high specificity. These nucleases rely on engineered protein domains that recognize and bind to specific DNA sequences, enabling targeted cleavage. While they lack the simplicity of CRISPR’s RNA-guided mechanism, they offer advantages in minimizing off-target effects. Studies have shown that ZFNs can achieve precise gene corrections in diseases such as hemophilia and sickle cell anemia, while TALENs have been used in preclinical models for conditions like Duchenne muscular dystrophy.

Mechanisms Of Cell-Specific Recognition

Achieving precise gene editing within living organisms depends on the ability to recognize and target specific cell types. Cell-specific recognition ensures that gene-editing components reach only the intended cells, minimizing off-target effects and reducing unintended genetic modifications. This process relies on interactions between engineered molecular components and unique cellular markers that differentiate one cell type from another.

One primary mechanism involves ligand-receptor interactions. Cells express distinct surface proteins that act as molecular signatures, allowing targeted binding by engineered ligands or antibodies. For example, researchers have exploited the transferrin receptor (TfR) for targeted gene editing in hematopoietic stem cells, as TfR is highly expressed on these cells. By conjugating gene-editing machinery to transferrin or TfR-specific antibodies, scientists enhance delivery specificity, ensuring that only hematopoietic stem cells undergo modification. Similar strategies have been used for neuronal targeting, utilizing ligands that bind to receptors such as low-density lipoprotein receptor-related protein 1 (LRP1), which is highly expressed in the central nervous system.

Another approach involves cell-specific promoters that restrict gene-editing activity to designated cell types. Promoters regulate gene expression, and incorporating cell-type-specific promoters into gene-editing constructs ensures that editing enzymes such as Cas9 or base editors are only expressed in the intended cells. For instance, the albumin promoter has been used for liver-specific expression of gene-editing components, enabling precise modifications in hepatocytes while preventing unwanted activity in other tissues. Similarly, the synapsin promoter has been leveraged for neuron-specific gene editing, ensuring that modifications occur exclusively within neuronal populations.

RNA-based targeting mechanisms add another layer of specificity. Certain gene-editing platforms utilize guide RNAs that are selectively processed or stabilized in specific cell types. MicroRNA (miRNA)-responsive elements have been incorporated into gene-editing constructs to exploit endogenous miRNA expression patterns. By including sequences complementary to miRNAs highly expressed in non-target cells, researchers can induce degradation of gene-editing components outside the intended population. This approach has been particularly useful in reducing off-target editing in tissues where gene modification is undesirable, improving both safety and precision.

Delivery Vectors For Gene Editing

Delivering gene-editing tools into living tissues requires specialized vectors capable of transporting molecular components across cellular barriers while maintaining efficiency and specificity. Researchers have explored a variety of delivery systems, each with unique advantages and limitations that shape their applicability for different diseases and tissue types.

Viral vectors have long been a preferred option due to their natural ability to enter cells and deliver genetic material. Adeno-associated viruses (AAVs) are particularly favored for in vivo gene editing, as they exhibit low immunogenicity and provide long-term expression of delivered components. Different AAV serotypes demonstrate varying tissue tropisms, allowing researchers to selectively target organs such as the liver (AAV8), muscle (AAV9), or central nervous system (AAV-PHP.B). However, the limited packaging capacity of AAV—approximately 4.7 kb—poses challenges for delivering larger gene-editing constructs like CRISPR-Cas9. To address this, dual-vector strategies have been developed, where the editing components are split across two AAVs and reassembled within the target cell.

Lipid nanoparticles (LNPs) have emerged as a promising non-viral alternative, particularly for delivering gene-editing tools in RNA form. LNPs encapsulate messenger RNA (mRNA) or ribonucleoprotein complexes, shielding them from degradation and facilitating cellular uptake via endocytosis. This approach has shown success in preclinical models for liver-directed gene editing, as hepatocytes efficiently internalize LNPs. Unlike viral vectors, LNPs do not integrate into the genome, reducing the risk of insertional mutagenesis. Their modular composition allows for surface modifications that enhance tissue targeting, such as the incorporation of N-acetylgalactosamine (GalNAc) ligands, which improve liver-specific delivery by binding to asialoglycoprotein receptors on hepatocytes.

Physical delivery methods provide another avenue for in vivo gene editing, leveraging techniques such as electroporation and hydrodynamic injection to introduce genetic material into cells. Electroporation applies brief electrical pulses to create transient pores in the cell membrane, facilitating the entry of gene-editing components. While commonly used for ex vivo modifications, advancements in microelectrode technology have enabled localized in vivo applications, particularly in tissues like the retina and skeletal muscle. Hydrodynamic injection involves the rapid infusion of a large volume of DNA or RNA solution into the bloodstream, creating temporary disruptions in endothelial barriers that allow genetic material to enter hepatocytes. Though effective for liver-targeted editing, this method requires precise control to minimize tissue damage and ensure reproducibility.

Role Of Proteins And Ribonucleoproteins

The effectiveness of in vivo gene editing depends on the precise activity of proteins and ribonucleoproteins (RNPs), which serve as the molecular machinery responsible for modifying DNA. These components must recognize target sequences with high fidelity and execute edits in a controlled and efficient manner. Their structural and biochemical properties determine specificity, stability, and functionality within the cellular environment, influencing both therapeutic outcomes and potential off-target effects.

Cas proteins, particularly Cas9 and Cas12, play a central role in CRISPR-based gene editing. Their ability to introduce targeted DNA breaks is dictated by guide RNA interactions, which ensure sequence-specific recognition. Structural studies have revealed that engineered variants, such as high-fidelity Cas9 (HF-Cas9) and enhanced specificity Cas9 (eSpCas9), exhibit reduced off-target cleavage by altering the enzyme’s conformational dynamics. Additionally, some Cas enzymes, such as Cas12a, process their own guide RNAs, eliminating the need for separate tracrRNA components and simplifying delivery strategies. These refinements enhance editing precision, making them valuable for therapeutic applications.

Beyond CRISPR-associated proteins, other nucleases such as ZFNs and TALENs rely on engineered protein-DNA interactions to achieve specificity. Their modular structure allows for precise targeting, but the complexity of protein design limits their scalability compared to RNA-guided systems. However, recent advancements in computational protein engineering have improved the efficiency of ZFNs, enabling their application in diseases that require highly controlled gene modifications.