Recent advances in the nanoplatform-mediated delivery of gene editing technologies to the lungs

Since the discovery of the CRISPR/Cas system as a powerful tool for genetic editing, it has been recognized as a promising therapeutic approach for diseases that are otherwise hardly manageable and curable [1]. The potential to permanently correct the underlying cause of a disease, rather than managing symptoms, has led to intensive research across numerous fields. In particular, gene editing has become an invaluable tool for the treatment of lung-related diseases.

A promising target for this technology is cystic fibrosis (CF). Base editing strategies can correct various mutations in the CF transmembrane conductance regulator (CFTR) gene, including multiple nonsense mutations, thereby restoring function. A study in CF mice has already shown a 50% correction rate in lung stem cells [2]. Another possible target is the SFTPC gene, in which gain-of-function mutations can cause interstitial lung disease. Experiments in mice have led to effective editing in almost 20% of the lung tissue [3]. Research into lung cancer treatment has gained considerable attention. The CRISPR/Cas system has been effectively used to edit PD-1 ex vivo in T cells to enhance immunological response and directly disrupt oncogenes to inhibit further tumor growth [4,5].

Although this system holds immense potential, its safe and effective delivery remains challenging. Several approaches have recently been investigated. At the initial stage, it must be determined in which form the CRISPR/Cas system will be delivered. The most direct method involves preassembling the Cas protein with the single guide RNA (sgRNA), which specifies the genomic target, to form the ribonucleoprotein (RNP) complex. An advantage is its short half-life, reducing possible off-target modifications [6]. However, the protein must undergo rigorous purification processes to be endotoxin-free, complicating production. Additionally, preexisting IgG antibodies against Cas can reduce its efficacy and elicit immune responses, posing safety concerns [7]. Another option is the delivery of plasmids encoding the Cas protein and sgRNA [4]. The requirement for prior transcription followed by translation leads to a temporal delay, which may increase the risk of an immune response and reduce transfection efficiency. Additionally, plasmids encoding both the Cas sequence and the sgRNA are commonly very large in size (80,000–100,000 bp), challenging their packaging, delivery and nuclear import [8]. A promising approach is the delivery of Cas messenger RNA (mRNA) along with the sgRNA. The mRNA is comparatively shorter than a plasmid (4,000 nt) and can be directly translated into the functional protein several times, reducing the overall amount of therapeutic needed for protein expression [8,9]. Inclusion of donor DNA templates allows for site-specific corrections [8]. Notably, in addition to nuclease-based agents like CRISPR/Cas, oligonucleotide-based agents can also be used for genome editing [8]. Irrespective of the approach, highly effective delivery systems are required, as cells do not readily take up proteins or nucleic acids.

Viral vectors are, by nature, highly effective in transfecting cells. As such, they have already been used to deliver CRISPR/Cas systems in vivo. However, they also bear the risk of immunogenicity and genome integration, leading to an overall shift toward non-viral alternatives [10].

Lipid-based vectors represent a widely used non-viral delivery platform. In recent years, lipid nanoparticles (LNPs) have elicited substantial interest from the scientific community. This is partly due to the FDA approval of the Covid-19 vaccines confirming the safety and efficacy of LNPs for the delivery of mRNA, as well as proving the scalability of the technology [11]. LNPs typically consist of four different lipids: the ionizable lipid, cholesterol, helper lipid, and PEGylated lipid, all of which are necessary for effective delivery [11]. However, the current composition of these lipids favors hepatic delivery, requiring further adaptations to drive tropism toward the lungs [12]. In 2020, Cheng et al. [12] released their landslide SORT paper. SORT, an acronym for Selective ORgan Targeting, uses the addition of another permanently charged lipid to target specific organs. Specifically for lung tropism, the addition of cationic lipids such as DOTAP and DDAB drives pulmonary targeting. It is believed that the cationic lipid leads to the formation of a different protein corona, facilitating tropism [12]. However, this technology also bears one major downside: thrombosis. Several papers have reported the appearance of thrombi as a direct result of lung-targeting LNPs in vivo. Although pretreatment with bivalirudin has inhibited the formation of thrombosis, this side effect limits therapeutic applicability [13]. Similarly, while cationic lipids can be effective, their use should be carefully controlled to reduce the risk of severe adverse reactions in vivo.

An alternative strategy to enhance lung tropism is to modify the chemistry of the ionizable lipid. Incorporating amide bonds in the linker structure instead of ester bonds has been shown to improve the lung targeting efficiency of LNPs [14]. Xu et al. [15] performed a high-throughput screening of 96 different cationic, degradable lipids within LNP formulations for lung targeting, rather than adding a separate SORT lipid. Their top-performing formulation outperformed a SORT-based LNP in in vivo gene editing [15].

Direct inhalation of the therapeutic offers local delivery and can help circumvent issues associated with systemic administration, such as toxicities caused by targeting and rapid clearance. As LNPs are already suspended in a buffer during manufacturing, nebulization for inhalation is a logical approach. However, this introduces new challenges. The shear stresses generated during aerosolization created by vibrating mesh nebulizers (VMN), which are necessary to generate an inhalable aerosol, cause the LNP to lose its payload and aggregate. These changes in the physicochemical parameters reduce bioactivity, requiring further formulation optimization [16]. Strategies to address this issue include the addition of viscosity enhancers and increasing the amount of PEGylated lipid [16,17]. Liu et al. presented an innovative approach by using charge-assisted stabilization to increase the repulsive forces between the LNPs during nebulization. By including negatively charged peptide-lipids into the formulation, they minimized payload loss and aggregation, leading to highly effective LNPs for in vivo applications [18]. Another option is to use alternative nebulization strategies that minimize shear stress [19]. LNPs can also be administered as a dry powder. However, the drying step introduces additional stresses, which require close monitoring of process parameters and formulation optimization [20].

For lung-targeting, direct pulmonary administration should be prioritized. Despite the shear stresses imparted by VMN, formulation optimization can preserve therapeutic efficacy. Further research on nebulizer technology is also necessary. While innovative nebulization devices have demonstrated promising results in preserving formulation efficacy, they still lack clinical approval. Therefore, optimizing formulations for widely used VMN or adjusting VMN operational parameters are currently more relevant for clinical translation.

Other non-viral vectors include polymeric delivery systems. Among the various types, biodegradable poly beta-amino esters (PBAEs) have emerged as versatile candidates for RNA delivery due to their high encapsulation efficiency and biocompatibility. PBAE-E63 has demonstrated effective lung targeting, making it a promising candidate if systemic delivery of gene editing components is aimed for [21]. The tunability and physicochemical stability of PBAEs also make them highly suitable for nebulization [22]. Other commonly used polymers include poly(amine-co-ester) (PACE), poly(lactic-co-glycolic acid) (PLGA), and polyethyleneimine (PEI) [23,24]. While these delivery systems have already been used for pulmonary delivery, their applicability in pulmonary gene editing remains underexplored.

The growing interest in LNPs for pulmonary delivery is reflected in ongoing clinical studies. ReCode Therapeutics has initiated two Phase 1 clinical trials for the inhaled delivery of mRNA encoding DNAI1 (RCT1100) and CFTR (RCT2100). Additionally, a gene editing formulation targeting CFTR is currently in the preclinical stage [25].

Pulmonary gene editing offers the possibility of curing previously untreatable diseases. In combination with the advances in delivery systems and a paradigm shift in regulatory agencies, this field is steadily moving toward clinical translation.

Funding Statement

This paper was not funded.

Authors contributions

Moritz Marschhofer: Conceptualization, manuscript writing, and revisions.

Simone P. Carneiro: Conceptualization, manuscript review, and revisions.

Olivia M. Merkel: Conceptualization, manuscript review, and revisions.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing assistance

ChatGPT was used to improve the readability and style of the manuscript. The authors take full responsibility for the content.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.