Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis

Nonspecific packaging

Expressing the desired cargo in producer cells is the most straightforward packaging strategy. Transfecting producer cells with plasmids encoding Cas9 and its cognate sgRNAs leads to the loading of functional Cas9-sgRNA RNPs into the EVs that are secreted either physiologically ^{60 –62} or due to the overexpression of exogenous factors that promote their budding and secretion, such as the viral fusogen vesicular stomatitis virus G glycoprotein (VSV-G). ^63,64 The exact mechanism of cargo loading has not been fully elucidated. Chen et al. showed that sgRNAs can be recruited into EVs even in the absence of the Cas9 protein, and vice versa, at least in part, suggesting a stochastic encapsidation, ⁶² whereas Lainšček et al. found that the loading of Cas9 derivates into EVs (named GEDEX) was boosted by the presence of sgRNAs. In the same work, addition of a membrane-targeting motif to Cas9 did not improve encapsidation, indicating an intrinsic affinity of Cas9-sgRNA RNPs for packaging into EVs. ⁶¹ This is in sharp contrast with the strategy proposed by Whitley et al., who fused a myristoylation signal to Cas9 to promote its membrane anchoring, and thus its packaging. ⁶⁵ Montagna et al. identified colocalization of the Cas9 protein and the sgRNA at the cell periphery as a limiting factor for Cas9-sgRNA RNP packaging. By placing the sgRNA transcription under the control of the cytosolic RNA polymerase of the bacteriophage T7—which is stably expressed by the producer cell line ⁶⁶ —instead of the more commonly used nuclear polymerase III, the investigators generated vesicles (named VEsiCas) that mediated editing levels comparable with direct transfection of Cas9- and sgRNA-encoding plasmids. ⁶³

Fusion of cargo proteins to structural viral proteins

In the first example of Cas9-VLP^HIV design, Cas9 was fused to the N-terminus of Gag^HIV through a HIV-1 PR-cleavable linker to ensure its release inside the capsid during particle maturation. In the same construct, a membrane-targeting protein domain was added to the Cas9-Gag fusion protein to promote its encapsulation. The sgRNA, however, was recruited inefficiently, and was therefore encoded in the lentiviral genome. ³⁶ Hamilton et al. reported that the fusion of Cas9 to the C-terminus of Gag^HIV allowed sufficient incorporation of Cas9-sgRNA RNPs to support robust editing, even in the absence of an sgRNA-expressing genome—possibly due to the C-terminal fusion allowing more efficient Cas9-sgRNA interactions compared with the N-terminal one (system named Cas9-EDVs). ³⁸ The same group has now shown that fusing Cas9 to a truncated Gag^HIV, containing only the MA domain and the C-terminus of CA, and expressing this fusion protein in producer cells that do not additionally express the native Gag-Pol^HIV yields particles that are 25% smaller and less complex than the original architecture (named mini-EDVs) but are equally or more efficient. ⁵⁴ Recently, Haldrup et al. designed novel Cas9-VLP^HIVs—termed lentivirus-derived nanoparticles (LVNPs)—in which Cas9 is fused to the C-terminus of a truncated Gag-Pol^HIV, and where sgRNAs are equipped with optimized scaffolds that increase their stability and promote their interaction with Cas9 to facilitate corecruitment. This architecture was also adapted to shuttle base editor and prime editor RNPs. ⁶⁷

The first all-in-one Cas9-VLP was the friend MLV-based design developed by Mangeot et al. (named Nanoblades) in which Cas9 was fused to the C-terminus of Gag^MLV and efficiently complexed with sgRNAs. ⁶⁸ Building upon this architecture, Banskota et al. developed a platform (named eVLPs) capable of delivering Cas9, the base editor ABE8e, ⁶⁹ and, in a more recent adaptation, the prime editor RNP as well—the latter a significantly larger cargo (∼250 kDa for the prime editor, ∼160 kDa for Cas9) that required the systematic identification of specific packaging bottlenecks. ⁷⁰ A considerable improvement came from switching to a Moloney MLV-based architecture. The better understanding of substrate specificity of the Moloney MLV PR enabled the inclusion of more efficient PR-cleavable sites in Gag^MLV-Cas9 linkers that enhanced Cas9 release upon particle maturation. ⁶⁹ Interestingly, this is in sharp contrast with the previously reported findings of Ngo et al. that most Cas9-VLPs do not complete maturation, and that uncleaved Gag^HIV-Cas9 is fully functional and localizes to the cell nucleus. This strongly suggests that, at least in this design, Gag^HIV-Cas9 is associated with the VLP membrane rather than the capsid core, supporting a model in which PR-mediated maturation is responsible for forming a highly structured capsid core in retroviruses and retroviral vectors, but is not required to generate VLPs. ⁵⁴

A crucial step in the architectural optimization of Cas9-VLPs is the titration of the Gag-cargo: Gag-Pol plasmid ratios in producer cells to ensure the ideal stoichiometry that maximizes cargo loading while efficiently supporting particle assembly. Indeed, Gag-Pol is widely believed to ensure the production of sufficient native structural polyprotein for efficient particle formation and egress while also supplying the PR, which is still considered necessary for the maturation of VLPs into functional particles according to the current consensus. ^67,69 Indikova et al. circumvented this issue by fusing Cas9 to the accessory HIV protein Vpr, and by equipping the fusion transcript, which is expressed in packaging cells, with the same regulatory elements that govern the spatiotemporal distribution of the Gag-Pol^HIV mRNA. ³⁷

Tethering cargo proteins to vesicle-enriched transmembrane proteins via dimerization modules

In contrast to VLPs, EVs lack a structured protein core, so their cargo is often anchored to vesicle-enriched transmembrane proteins. However, due to the large size of RNPs, direct fusion would likely hinder the function and folding of both proteins. Anchoring is therefore typically mediated by dimerization modules—which additionally ensure the reversibility of the assembly.

Wang et al. first applied this principle to arrestin domain-containing protein 1 (ARRDC1)-mediated MVs, or ARMMs—a subtype of small MVs (∼50 nm) whose budding from the plasma membrane is mediated by ARRDC1. They exploited the specific interaction of AARDC1 with the WW protein domain—a 40-amino acid module characterized by two conserved tryptophans (W)—by fusing WW to Cas9, which they demonstrated did not impair its enzymatic activity. ⁷¹ This delivery vector has now been refined into a patented platform for tissue-specific in vivo delivery of gene editor RNPs. ⁷²

Zhang et al. devised a similar strategy to encase protein cargo in a broader spectrum of MVs by exploiting two self-associating fragments of a split-GFP system ⁷³ to tether Cas9 to the cytoplasmic end of VSV-G and thus promote its active loading in VSV-G-containing MVs (named gectosomes). ⁷⁴ Similarly, Ye at al. leveraged the affinity between GFP and a GFP-targeting nanobody to tether Cas9 to the tetraspanin CD63—a well-known exosomal marker ¹⁹ —to produce exosomes enriched in functional Cas9-sgRNA RNPs (named M-CRISPR-Cas9 exosomes). ⁷⁵ An equivalent approach based on the P3/P4 coiled coil peptides—a pair of α-helical peptides that selectively and strongly bind to each other—was proposed by An et al. to tether prime editor RNPs to Gag in VLP^MLVs. ⁷⁰

A tighter control on particle production can be introduced by governing cargo loading with an inducible dimerization system. Campbell et al. first demonstrated this by anchoring the FK506-binding protein 12 (FKBP12, also known as DmrA) to the cell membrane through myristoylation, and by fusing the T82L mutant FKBP12-rapamycin binding domain (FRB, also known as DmrC) to Cas9. Addition of a rapamycin analogue triggers DmrA and DmrC to dimerize in producer cells, thus promoting loading of the Cas9-sgRNA RNPs in secreted EVs (named gesicles). Once inside the target cells, rapamycin becomes diluted, prompting cargo release. ⁷⁶ This is in line with strategies proposed by Ilahibaks and Ardisasmita et al. ⁷⁷ (named Cas9-TOP-EVs) as well as by Gee et al., ⁷⁸ (named NanoMEDIC), which were developed based on this principle. Gee et al. fused FKBP12 to Gag^HIV to promote cell membrane localization of the viral protein and equipped the sgRNA with the HIV Ψ packaging signal to enable its Gag-mediated recruitment into the vesicle. The sgRNA is transcribed in the producer cells from the HIV long-terminal repeat promoter and subsequently released from the viral RNA through the self-cleavage of two flanking ribozymes. This combined approach was shown to maximize Cas9 and sgRNA recruitment into the VLP. ⁷⁸

Based on reports that rapamycin-induced heterodimerization may not be fully reversible, ⁷⁹ Stranford et al. developed a similar system based on the plant hormone abscisic acid-induced dimerization between a truncated form of the abscisic acid insensitive protein 1, which was fused to Cas9, and a pyrabactin resistance-like protein, which was tethered to an antibody single-chain variable fragment (scFv) protein embedded in the EV membrane. ⁸⁰ Cas9-sgRNA RNPs have been encapsulated in EVs also by exploiting the blue light-dependent heterodimerization of the cryptochrome 2 (CRY2) and its ligand protein CIBN, which can be enriched on the plasma membrane of secreted vesicles by fusing it either to an EV marker such as CD9 or to a membrane-anchoring peptide. ^81,82

sgRNA-mediated recruitment

Most Cas9-sgRNA RNPs loading strategies focus on active recruitment of Cas9 while relying on the intrinsic affinity of Cas9 for sgRNAs to encase functional RNPs into CDVs. Conversely, Lyu et al. designed an sgRNA-mediated approach to load Cas9-sgRNA RNPs in VLP^HIVs by replacing the sgRNA tetraloop with the com RNA aptamer—a phage-derived sequence characterized by a secondary hairpin structure—and fusing Gag^HIV to the aptamer-binding protein COM, which selectively and specifically binds the com hairpin. ^83,84 This system (named LVLPs) was originally developed to deliver SaCas9, but was later adapted to shuttle both SpCas9 ⁸⁵ and ABE ⁸⁶ RNPs. The same group was also able to enrich such cargos in EVs by fusing COM to CD63. This was shown to increase the average vesicle size without a significant impact on morphology or yield. ⁸⁷

Chemical/physical methods

Wan et al. were the first to electroporate a cargo as large as the Cas9-sgRNA RNP into exosomes purified from cultured cells, which resulted in a highly transient (half-life <3 h) and nonimmunogenic delivery vehicle (named Exosome^RNP). ⁸⁸ More recently, Mun et al. applied the same technique to EVs isolated from human serum (named EV@RNP). ⁸⁹ Both studies reported that electroporation did not alter the morphology and size distribution of the particles. ^88,89 Consistent with this, Majeau et al. reported comparable results using protein transfection with the Lipofectamine CRISPRMAX reagent. ⁹⁰ In Busatto et al., Cas9-sgRNA RNPs were incubated with cationic lipids to form synthetic LNPs. These could then be hybridized with exosomes resulting in only slight changes in the mean size and the zeta potential of the exosomes ⁹¹ —the latter being an indicator of surface charge and colloidal stability. ⁹² A similar technique was leveraged by Zhou et al. to convert the RNA-targeting nuclease Cas13a into a detection system for cancer-associated miRNAs in circulating exosomes. ⁹³

As final note, it is crucial to emphasize that the CDV architectures discussed in this section were designed to target distinct genomic loci in various in vitro or in vivo models. Therefore, it is difficult to compare efficacy outcomes or direct performances between delivery systems (Fig. 2; Table 1).

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Figure 2.

Methods for loading clustered regularly interspaced short palindromic repeats (CRISPR)-Cas cargoes into cell-derived vesicles (CDVs). The figure illustrates the methods for cargo loading used in the Cas9-CDV systems that are discussed in Chapter 3 and referenced in Table 1 using the same color scheme. Nonspecific packaging (light blue): The Cas9 protein and sgRNAs are coexpressed in the producer cell line, usually via plasmid transfection. They assemble into Cas9-sgRNA RNPs due to their intrinsic affinity and are packaged into budding CDVs via stochastic, nonspecific interactions. Fusion to viral structural protein (green): Cas9 is fused to the precursor of a viral structural protein—most commonly Gag. Coexpression of the Gag-Cas9 fusion protein together with the sgRNA and the native Gag-Pol polyprotein allows the assembly of VLPs that encapsulate Cas9-sgRNA RNPs. Dimerization with transmembrane protein (yellow): Cas9-sgRNA RNPs are tethered to plasma-membrane proteins enriched on the EVs via dimerization modules. One of the dimerizing proteins (DM) is fused to the cytosolic domain of the EV-enriched transmembrane protein, whereas its binding partner is fused to Cas9. The dimerization may be constitutive or triggered by small molecules or photons. sgRNA-mediated recruitment (pink): the Cas9-sgRNA RNPs are tethered to the membrane of budding CDVs by exploiting the affinity between an aptamer-binding protein (ABP) and an RNA aptamer sequence. The ABP is fused to a membrane-tethered, CDV-enriched protein—either a viral structural protein or a transmembrane EV marker protein—while the sgRNA is modified to contain the aptamer sequence recognized by the ABP. Postisolation loading (indigo): The Cas9-sgRNA RNPs are assembled in vitro and loaded into isolated EVs via chemical or physical disruption of their membranes—for example, by electroporation or protein transfection.

Clarification

Clarification involves the removal of coarse contaminants, such as cell debris or intact cells—the latter being particularly relevant for producer cell lines grown in suspension, which is becoming the standard practice in the industry. Clarification is usually achieved through low-speed centrifugation, followed by microfiltration ^97,99,100 (pore size 0.1–1 μm). ¹⁰¹

Intermediate purification

Intermediate purification eliminates impurities such as salts, serum components, and macromolecules produced by the packaging cells. It is usually coupled with a concentration step to reduce the particles’ suspension volume—which is crucial to achieve sufficient in vivo potency. ^100,102

Proteins and nucleic acids are particularly challenging contaminants. Host cell proteins (HCPs) derived from the packaging cells or from serum ^97,100 can form aggregates with similar size and density as the desired products. ¹⁰¹ DNA originating from the genome of packaging cells or from the plasmids used to transfect them tends to copurify with retroviral particles due to its high molecular weight and negative charge—which are similar to those of proteoglycans. While digestive enzymes can effectively eliminate these contaminants, their removal requires a supplementary purification step. ^97,100,103 Alternatively, contaminants can be depleted upstream by implementing earlier harvest times, which minimizes dead cells, ^99,100 and by using serum-free culture medium, ^97,103 which also reduces costs, interbatch variability, and microbial contamination risks. ¹⁰² All of these strategies, however, can hinder particle yield. ^99,104 The removal of cosecreted vesicles and defective particles—that is, particles carrying incomplete cargos—is more demanding ⁹⁸ and further complicated by the fact that CDV-producer cells often exhibit a general increase in the secretion of vesicles with altered lipid composition and size distribution profile after being transfected. ^{105 –107} Techniques to enrich the yield of full capsids over empty and partially full ones have been developed for AAVs, but not for enveloped viral vectors. ⁹⁸ Protocols for efficient production of VLPs lag even further behind, partly due to their internal configuration being less well understood to date—as recently highlighted by Hamilton et al. ¹⁰⁸

Polishing

Lastly, polishing is the removal of remaining impurity traces, which is often performed through size-exclusion chromatography (SEC). This ensures high particle recovery and activity preservation but results in an undesirable dilution of the final product. Multimodal chromatography, which combines SEC and AEX, has also been applied successfully for polishing. ^94,99,100 The final stage for all clinical-grade materials is sterile filtration, which reduces yields due to nonspecific particle-filter interactions, along with the addition of excipients to preserve biological activity and product stability through freezing. ^94,95,99 The unavoidable particle loss is compensated by a reduced activation of proinflammatory pathways, which is likely to improve editing efficiencies and target cell viability. ¹⁰³

Nonfunctional titration of Cas9-CDVs

At the laboratory scale, the quality of CDVs is usually assessed through nonfunctional titration methods such as immunoblotting, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, which analyze the bulk population of produced particles and quantify them by measuring cargo proteins and envelope markers—or, in the case of VLPs, capsid components—as well as detecting contaminating proteins. These approaches have several limitations. First, they cannot distinguish between vesicle-associated and free proteins, thus not providing insight on the actual localization of either contaminants or cargo. Second, due to the lack of suitable CDV-specific markers, these techniques cannot analyze the entire secretome nor isolate a specific vesicle subpopulation. Finally, they do not provide accurate insight on the internal particle organization.

Nonetheless, ELISA is the most frequently used strategy to estimate the efficiency of Cas9 loading in VLP preparations. Following the downstream processing, antibodies directed either against Cas9 or an embedded peptide tag are used to estimate the concentration of gene editor proteins in a VLP sample. ^{36,69,70,127 –131} Similarly, the concentration of viral particles is derived from the concentration of the CA protein, the most abundant retroviral protein—p24 for VLP^HIVs ^{36,38,67,84,132,133} and p30 for VLP^MLVs. ^69,70 Importantly, this is not a direct quantification but rather an estimation based on the assumption that individual VLP^HIV and VLP^MLV particles contain ∼2000 p24 molecules ¹³⁴ and ∼1800 p30 molecules, ¹³⁵ respectively. For VLP^HIVs, commercial kits ensure the anti-p24 antibodies solely bind to particle-associated p24 molecules. For VLP^MLVs, ∼20% of the total p30 molecules detected in solution are assumed to be particle associated. ¹³⁵ Together, this allows to estimate the average number of Cas9 molecules per particle. PCR quantification of viral genomes, a key method for titrating viral vectors, is not suitable for Cas9-CDVs because a viral genome is usually lacking. However, reverse transcription quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR) can estimate the average number of sgRNAs per particle. ^96,98,136

It is worth noting that all protein- and RNA-based approaches for CDVs titration are physical titration methods: they quantify total particle counts without assessing their integrity and potency and are therefore incapable of discriminating between functional and nonfunctional particles.

Functional titration of Cas9-CDVs

In contrast to physical titration, functional titration methods quantify only those particles capable of delivering complete and functional cargo. In the case of Cas9-CDVs, the readout for cargo functionality is the efficiency of genome editing. However, this measurement is heavily influenced by several factors that are not directly related to the CDV architecture, including the type of gene editor used, the targeted genomic location, and the type of target cell (either cultured or in vivo). These factors introduce cargo-specific variations in editing efficiencies, complicating direct comparisons between CDVs that carry different gene editor RNPs.

Cargo-specific biases in functional titration can be minimized by implementing standardized reporter systems, where each CDV shuttles the same gene editing machinery, targets the same genomic site, and is tested in the same cell type using a consistent methodology to measure editing efficiency. This approach ensures that observed differences in genome editing efficiencies between different CDV architectures are attributable solely to variations in delivery efficiency. Reporter systems can be established in increasingly complex models, ranging from cell lines to primary cells and even organoids and organ-on-chip platforms. In addition, this approach would provide a valuable tool to monitor the consistency between batches of the same Cas9-CDV product manufactured at different times, thereby supporting the scalability and reliability of these delivery vehicles and helping to narrow the gap between preclinical research and clinical applications of Cas9-CDVs as advanced therapeutic medicinal products. Specifically, genome-editing medicinal products (GEMPs) are an area where accurately estimating the numbers of functional versus nonfunctional particles in a sample is essential. ¹³⁷ Potency assessment should directly relate to the intended biological function (defined as on-target efficiency). Establishing a suitable potency assay, predictive of biological activity, during the early stages of product development is key to ensure comparability between batches of the same product and between different product versions in preclinical studies, as well as to demonstrate product consistency during clinical testing. ¹³⁸ The recent approval of Casgevy by the European Medical Agency is an important milestone that will provide further regulatory guidance for GEMPs. ¹³⁹

Analytical techniques to assess Cas9-CDVs

The use of CDVs as delivery vehicles in gene therapy is limited by our incomplete understanding of their biology, the lack of uniform standards for their production, and the absence of straightforward, cost-effective methods to assess their functionality. As a result, CDVs are still a “black box” very far from meeting the rigorous quality standards imposed on clinical-grade materials. To advance the use of CDVs in GEMPs, we propose that advanced techniques for particle characterization can be imported from the fields of viral vector production, VLP vaccine manufacturing, and EV-based diagnostics. Although these approaches cannot address all currently unmet needs, they can undoubtedly serve as driving force in the clinical advancement of Cas9-CVD applications.

Limitations of current analytical techniques

Choosing the optimal method is challenging, as each excels for a different particle size range, and all have an unclear limit of detection. Technique-specific biases as well as varying abilities to resolve aggregates and exclude contaminants can cause significant discrepancies in particle concentration and size distribution—making the use of multiple orthogonal methods recommended. ^96,136 Moreover, the uptake kinetics of any vesicle are influenced by both its size and surface charge ^141,145,146 —an often overlooked parameter in quality assessments. The zeta potential can be assessed using electroacoustic ¹⁴⁷ and electrophoretic ¹⁴⁵ techniques, specialized NTA protocols, ¹⁴⁸ or tunable resistive pulse sensing. ^96,98

The biomolecular corona of CDVs is perhaps as important as its size and charge, but it is not frequently considered when characterizing the molecular properties of vesicles. The corona is a dynamic layer of macromolecules that rapidly adsorb—that is, adhere to the surface—onto nanoparticles upon contact with biological fluids. This process is driven by biomolecule–nanoparticle and biomolecule–biomolecule interactions. The protein corona is a key determinant of a vesicle’s biological identity and tropism. ^{149 –151} Recent studies have used mass spectrometry to investigate the corona formed on inorganic and biomimetic nanoparticles, including liposomes, in human plasma. ^149,152,153 Similar analyses have been conducted for enveloped viruses ¹⁵⁴ and circulating EVs, where an additional challenge lies in distinguishing macromolecules that are part of the vesicle from those associated with its corona despite their shared biological origin. ^151,155 To our knowledge, the corona of CDVs used in drug delivery following in vivo administration has not yet been characterized. Some evidence suggests that the corona starts forming during particle production, raising the possibility that some macromolecules currently considered contaminants may actually be parts of the vesicle’s identity. ^96,151

Finally, another understudied aspect in the quality assessment of CDVs is the posttranslational modification status of their structural proteins. Glycosylation, in particular, is gaining recognition as a key determinant of protein function and stability in several biological processes. For example, N- and O-linked glycosylation of viral and host cell proteins is an important tool through which certain viruses, including influenza, HIVs, and coronaviruses, modulate protein folding and receptor binding. Analytical methods to examine and interrogate these modifications, however, have not yet been developed (Fig. 3). ^{156 –158}

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Figure 3.

Downstream processing and quality control of Cas9-CDVs. The figure outlines the fundamental steps of the downstream processing of Cas9-CDVs, from harvesting the particle-enriched medium to the three purification phases (clarification, intermediate purification, and polishing), followed by final formulation for long-term storage. A final quality assessment step for the purified particles is included. For each step in the pipeline, the most used techniques are highlighted. Various alternative techniques for intermediate purification and quality control are presented, along with a schematic overview of their strength and limitations. Depending on the intended application, a single technique or a combination of methods may be used at each stage. The cryo-electron microscopy (EM) images are reproduced from Raguram et al. ¹⁴⁰ and is used here without alterations under a Creative Commons license (http://creativecommons.org/licenses/by/4.0/).

Receptor binding, fusion routes, and cell entry

Similarly to how EVs and enveloped viruses—and, consequently, their derived vectors and VLPs—share similarities in their biogenesis pathways, they are also known to rely on similar mechanisms for cell entry.

The ability of a virus, along with its derived viral vectors and VLPs, to target specific cell types is defined as tropism. For enveloped viruses, this is determined by the affinity between host cell-type-specific receptors and the glycoproteins embedded in the viral envelope.

The binding of viral envelope proteins to cell receptors is a necessary first step in effective transduction of the target cells—meaning that only cells expressing these target receptors are permissive to viral entry. Once the viral particle is tethered to its cognate receptor, cell entry and thus cargo delivery may occur through one of two principal mechanisms: (i) direct fusion of the viral envelope with the target cell plasma membrane or (ii) internalization via endosomes and subsequent pH-dependent escape—the latter being more common than the former. In fact, the large majority of viruses—including VSVs, from which the most widely used envelope glycoprotein VSV-G is derived—rely on the endocytic machinery of the target cells to “hitch a ride” on the cytoskeleton from the plasma membrane to the nuclear periphery. This eases the passage through the crowded cytoplasm and minimizes the viral components presented at the plasma membrane, thereby delaying antibody or immune cell recognition. ^{162 –164} There are, however, notable exceptions to this general rule: Some members of the Retroviridae family, including HIV, as well as the Herpesviridae and Paramyxoviridae families, ^{165 –167} can directly fuse with the plasma membrane through a pH-independent entry mechanism that relies on specific conformational changes in the envelope protein upon receptor binding. This allows them to release their contents into the target cell cytoplasm, either as their primary entry strategy or as an additional route alongside endocytosis, depending on the target cell type.

While the molecular interactions of some viruses are well understood, they remain elusive for many others. However, as virology shifts from cultured cell models to in vivo experiments, it becomes evident that viruses exhibit significant redundancy and adaptability in receptor usage, endocytic pathways, penetration sites, and uncoating mechanisms. ¹⁶³ Of note, receptor-independent, nonspecific uptake pathways have also been described for both MLV and HIV virions ¹⁶⁸ —an important consideration when evaluating the specificity and safety of CDV-based strategies for the targeted delivery of gene editing tools.

Endosomal escape mechanisms

The endosomal–lysosomal system is a dynamic cellular network responsible for recycling, sorting, and processing internalized cargo. Following uptake via endocytosis, vesicles enter early endosomes—the cell’s primary sorting precinct. Here, depending on factors such as membrane composition, pH gradients within the early endosome, and the presence of molecular tags (e.g., ubiquitin), the vesicles will be either returned to the plasma membrane or directed toward the late endosomes, which subsequently fuse with lysosomes for degradation. To avoid being degraded in the host’s hydrolytic catabolic process, CDVs must escape from the endosome before its fusion with the lysosome. To achieve this, viruses and VLPs can co-opt some of the routes to prevent endosomal entrapment that are used by EVs, whose role as mediators of intercellular communication requires them to deliver intact molecular cargo to target cells and compartments.

Four distinct escape mechanisms have been proposed: membrane fusion, pore formation, back fusion, and pH buffering. ^{169 –173}

Nuclear import of cargo

It is currently unclear whether enveloped vehicles with a structured virus-like capsid, such as VLPs, can more effectively support directed transport of RNPs through the cytoplasm and into the nucleus compared with enveloped vehicles without a viral core, such as EVs. Nuclear entry of the viral capsid core, for example, in the case of HIV, has been debated for years, and current findings suggest that the core, at least under some circumstances, is transported through the nuclear pores before its disassembly within the nucleoplasm. ^182,183 Docking of the core at the nuclear pore complex is mediated by interactions between the capsid and cellular factors, leading to nuclear entry of an intact core. Hence, recent findings suggest that HIV nuclear capsids are intact or nearly intact until they uncoat immediately before integration of viral DNA. ¹⁸⁴ A certain structural elasticity of the core is thought to enable its nuclear entry. ¹⁸⁵

Whether such mechanisms also support delivery of Cas9-sgRNA RNPs by VLP^HIVs, such as LVNPs, and may promote unloading of genome-engineering proteins near open chromatin is currently unknown and needs further scrutiny. Likewise, further studies are needed to identify the cellular factors on which VLPs may rely to successfully deliver proteins—potentially uncovering variations between cell types and delivery systems that could impact efficacy.

Interestingly, the potential nuclear translocation of soluble and possibly even membranous EV components has also been suggested, for example, by the presence of the nuclear transport receptor karyopherin within EV proteome analyses. ¹⁸⁶

Tropism and pseudotyping

Pseudotyping involves altering the surface proteins of a viral particle or VLP by replacing its native envelope protein with a different targeting moiety. This changes the tropism of the particle, expanding or narrowing its ability to target specific cells—making this a crucial strategy for enhancing targeting specificity. ¹⁸⁷

One of the most widely adopted envelope proteins is VSV-G, which is characterized by high structural stability and very broad tropism. VSV-G, in fact, targets the members of the low-density lipoprotein receptor family, which are expressed in most human cells and tissues. ¹⁸⁸ To achieve targeted delivery to specific cells, naturally occurring envelope glycoproteins from other enveloped viruses with distinct tropisms can be leveraged. ¹²⁷ These include the envelope proteins of gamma-retroviruses (e.g., MLV), ¹⁸⁹ baboon retroviruses, ¹⁹⁰ and paramyxoviruses. ^191,192 Dual pseudotyping—that is, adding two envelope proteins to a particle—has also been demonstrated to provide specific advantages. For example, dual pseudotyping of LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoprotein significantly improved transduction of human cells, including hematopoietic stem cells. The unexpected synergy between these two elements stemmed from VSV-G facilitating the incorporation of wild-type SeV-HN into LVs, while SeV-HN cleaved the sialic acid on the VSV-G structure, ultimately enhancing transduction efficiency. ¹⁹³

Similarly, EVs must first bind to their target cells to deliver their cargo. A significant challenge hindering their therapeutic application is ensuring precise delivery to the desired tissues while avoiding accumulation at off-target sites. EVs are known to exhibit preferential affinity toward certain cell types, depending on a plethora of factors such as their surface protein profile, lipid and glycan composition, and charge (reviewed in Murphy et al. ¹⁹⁴ ), as well as cell type of origin. ¹⁹⁵ These factors can be harnessed and optimized toward cell-type-specific delivery of gene editor RNPs. In addition, similarly to viral vectors and VLPs, EVs can be customized by expressing viral glycoproteins or other recombinant fusion proteins on their surface via genetic modification of producer cells. ^196,197 VSV-G, for example, was found to enhance the delivery efficiency of EVs without causing significant changes to their size, morphology, and composition. ¹⁹⁸ Despite recent progress, many aspects of EV biology remain unclear. While the biodistribution and cell targeting of EVs can be modified, the uptake pathways that internalize them into cells are diverse and the mechanism can vary by cell and EV type. As is the case for viral particles, nonspecific uptake routes can limit the safety and efficacy of EV-based therapies. For both EVs and VLPs, endosomal entrapment remains a significant bottleneck, highlighting the need for new tools to enhance cargo release.

It is important to underscore that pseudotyping does not solely alter the tropism of a particle but also influences its biodistribution kinetics and mechanisms for entry and cargo release. The internalization dynamics and turnover rates of the target receptor can also impact these processes ¹⁹⁹ —making careful selection of the target receptor essential when designing a retargeting strategy.

Novel retargeting strategies

In addition to using naturally occurring viral envelopes, envelope proteins can be engineered or replaced with synthetic constructs to enhance cell-type specificity and thus improve safety by minimizing binding to off-target cells. The use of specific ligands to redirect delivery vehicles toward a specific target is known as ligand-directed targeting display.

To achieve this, the particles are first modified with a targeting ligand that binds with high affinity to a chosen receptor on the target cell surface. This binding mediates attachment but is not sufficient to initiate entry. Then, proteins that promote cell entry by facilitating membrane fusion, such as viral glycoproteins, are displayed on the surface of the particle. However, the intrinsic affinity of these proteins for their own canonical receptor could mediate cargo delivery to nontherapeutically relevant cell types. To prevent this, the natural receptor-binding properties of fusion protein should be inactivated via mutagenesis. ^{199 –201} This strategy has proven to be particularly difficult for viral glycoproteins that combine receptor binding and membrane fusion features in a single polypeptide, as is the case for VSV-G. Initial experiments using a membrane-anchored targeting moiety combined with a truncated VSV-G in LVs showed efficient delivery to the intended target cells, ²⁰² but nontarget cells were also transduced. ²⁰³ In contrast, some viruses, such as alpha-viruses (e.g., SINV) and paramyxoviruses (e.g., MV, TPMV, NiV), have separate polypeptides for receptor attachment and membrane fusion (reviewed by Perrin et al. ¹⁸¹ ). Engineering or replacing the receptor attachment polypeptide with specific targeting moieties, while maintaining the fusion protein unmodified, could provide a more straightforward approach to achieve cell-type-specific cargo delivery—although the existence of complex interactions between the receptor-binding and fusion glycoproteins may add a different layer of complexity to this strategy. ^165,204,205

One strategy for glycoprotein engineering is the production of tailored chimeric proteins that combine targeting domains from different viral proteins, creating hybrid envelopes that can enable more efficient and selective pseudotyping. For example, fusing the internal domains of the MLV glycoprotein to the target-binding domain of a codon optimized variant of the gibbon ape leukemia virus envelope protein enabled transduction of human primary B and T lymphocytes. ²⁰⁶

A different approach, which can further increase targeting specificity, leverages single-chain variable fragment antibodies (scFvs) against specific cell-associated antigens fused to the membrane-anchoring domain of an envelope protein. ²⁰⁷ For example, Hamilton et al. used scFvs against several CD antigens, fused to the transmembrane domain of CD8a and coexpressed with a mutant VSV-G, to selectively and specifically retarget their VLP^HIV design (termed EDVs) toward distinct subpopulations of human T cells. ¹⁰⁸ Recently, Strebinger and colleagues developed DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design), a modular platform that offers several immobilization strategies (i.e., protein A/G and SNAP-tag®) to stably display scFvs or full-length antibodies on the surface of LVs and VLP^MLVs, which is compatible with in vivo applications. ¹⁹⁹ In parallel, Stranford et al. developed GEMINI (Genetically Encoded Multifunctional Integrated Nanovesicles), a suite of methods for the production of genetically engineered EVs that display an anti-CD2 scFv fused to the C1C2 lactadherin domain scaffold for membrane tethering, in combination with the measles H/F glycoproteins to promote fusion. ⁸⁰

An alternative to scFvs is designed ankyrin repeat proteins (DARPins). Large DARPins libraries can be easily produced and screened to identify high-affinity binding partners for any given target molecule of interest. DARPins are less prone to aggregation compared with certain scFvs and enabled higher transduction efficiencies compared with scFvs directed toward the same target when fused to the measles H glycoprotein. ²⁰⁸ The use of the SpyCatcher-SpyTag chemistry—where a SpyTag inserted into an engineered Sindbis virus envelope protein serves as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher ²⁰⁹ —as well as split-intein systems that exploit the extremely rapid, high-affinity binding between the two halves of an intein catalytic site, ²¹⁰ has been described for tethering cell-targeting ligands to the envelope of delivery vehicles.

Most of the research on CDV pseudotyping has focused on protein-based strategies. However, other innovative approaches are emerging, such as the use of nucleic acid aptamers, which can be designed to bind to cell-specific target molecules and can be tethered to membranes through fusion with cholesterol or other membrane lipids. ^{211 –213}

Finally, the introduction of click-chemistry—a group of reactions involving the conjugation of molecules in a modular manner—has opened a wealth of options for the postisolation surface functionalization of vesicles. This method has proven to be robust, rapid, and easily scalable, ^194,214,215 and it has been reported not to induce significant changes in vesicle size and properties. ^216,217

This overview provides a general summary of available strategies for CVD retargeting; for a more comprehensive exploration, refer to He et al. ²⁰¹ and Gutierrez-Guerrero et al. ¹²⁷ (Fig. 4).

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Figure 4.

Methods for retargeting of CDVs. The figure depicts the most common retargeting strategies used to define the tropism of CDVs, as discussed in Chapter 4. In this illustration, a viral glycoprotein that combines receptor binding and membrane fusion features in a single polypeptide is shown—as is the case for the most commonly used viral envelope, vesicular stomatitis virus glycoprotein (VSV-G). Native envelope: Displaying a native envelope glycoprotein on the membrane of a CDV will confer to the latter the tropism of the virus from which the envelope is originally derived. Mutant envelope: Mutation of key amino acids in the target receptor recognition domain of an envelope glycoprotein may yield a mutant receptor that no longer binds its native target receptor but can still mediate the fusion between the viral envelope and cellular phospholipid bilayers—either the plasma membrane or the endosomal membrane, depending on the entry route of the virus from which the envelope glycoprotein is derived. Antibody-derived and antibody-mimetic targeting moieties: CDVs can be redirected toward specific targets by leveraging the affinity between a receptor of interest and a targeting moiety that derives from or mimics an antibody. Commonly used targeting moieties are antibodies (Abs), single-chain variable fragments (scFvs), nanobodies (Nbs), and designed ankyrin repeat proteins (DARPins). To ensure anchoring and correct localization on the plasma membrane, the targeting moieties are tethered to a transmembrane domain. This tethering can be direct, via fusion of the targeting moiety to the transmembrane domain, or indirect, via fusion of a docking site for the targeting moiety (Ab binder) to the transmembrane domain followed by incubation of the targeting moiety with the isolated CDVs in the appropriate conditions to promote stable binding to the docking site. While the targeting moiety dictates the tropism of the CDV, fusion potential is provided by coexpressing a fusion-competent mutant envelope protein.

Immune responses to the vector components

Due to their structure and origin, VLPs are expected to share at least some of the immunogenic features of the retroviral vectors from which they originate. For example, certain interferon-induced transmembrane proteins located on the plasma membrane as well as the endosomal membrane are known to restrict the cell entry of several viruses and viral vectors, including the ones of retroviral origin, presumably by reducing membrane fluidity upon recognition of several viral envelope glycoproteins ^218,219 —including VSV-G. ²²⁰ In addition, certain members of the Toll-like receptor (TLR) family have also been shown to restrict retroviral entry upon envelope glycoprotein sensing via activation of the NF-kß-dependent transcriptional program and consequent release of proinflammatory cytokines that stimulate the chemotaxis of phagocytes toward the site of infection. ^218,221,222 This suggests that concerns related to innate antiviral immunity may extend to EVs decorated with viral glycoproteins as well.

If the retroviral vectors overcome this first line of defense and reach the cellular cytosol, another array of innate immune responses can then be elicited by the retroviral capsid protein components. For instance, binding of the E3 ubiquitin ligase TRIM5α onto the retroviral capsid has a destabilizing effect on the latter, resulting in dysregulation of its uncoating and promotion of its proteasomal degradation—thus preventing reverse transcription of the retroviral vector-encoded transgene—as well as release of inflammatory mediators through the activation of the NF-kß and AP-1 transcription factors. ^218,223 In parallel, retroviral capsid proteins can also be sensed by certain TLRs, eliciting robust NF-kß activation and secretion of type 1 interferons (IFNs-1). ^218,222

The RNA-encoded transgenes packaged by retroviral vectors are shielded from the host immune system by the capsid and nucleocapsid structures until the reverse transcription step, during which they are first exposed to the cellular cytoplasm as uncoating occurs—although, as previously discussed, direct nuclear import of the assembled capsid has also been reported in certain conditions. ^182,183 The resulting double-stranded DNA is imported into the nucleus, where it integrates into the host genome. While for native retroviruses the integrated viral DNA is transcribed into new viral RNAs, which serve as retroviral genomes and as templates for the synthesis of retroviral proteins, ⁴³ in the case of single-round retroviral vectors, expression of the integrated transgene only gives rise to the product of interest. Retroviral nucleic acids are a major source of pathogen-associated molecular patterns that can be recognized by cellular pattern recognition receptors to stimulate innate immune responses. The viral RNAs, as well as retroviral vector-encoded transgenes, can be sensed both by TLRs located in the endosomal compartment and by cytosolic RNA helicases belonging to the retinoic acid-inducible gene I-like (RIG-I-like) receptor family, resulting in the activation of NF-kß and in the secretion of IFNs-1 and proinflammatory cytokines. A wide range of specialized receptors, including members of the TLR family, IFN-inducible proteins, and helicases, are able to specifically detect the reverse-transcribed viral DNA or retroviral vector-encoded transgene, along with all the intermediates formed during reverse transcription (e.g., RNA-DNA hybrids). Particularly notable among these receptors is cGAS, a cytosolic cGMP-AMP synthase that acts as the major sensor of retroviral DNA: the products of its enzymatic activity can activate the cGAS/STING signaling cascade, resulting in the induction of IFNs and IFN-stimulated genes—which include antiretroviral restriction factors such as APOBEC3G, a cytidine deaminase that mutates nascent viral DNAs during reverse transcription, TRIM5α, the aforementioned capsid proteins receptor, tetherin, a lipid raft-associated protein that can prevent the egress of newly assembled virions, and SAMHD1, which inhibits reverse transcription by depleting the dNTP pool. Such responses may affect cell function and, in some cases, even trigger apoptosis.

It is worth noting that, while native retroviruses are equipped with accessory proteins that can counteract or mitigate the effect of host restriction factors, these are generally omitted in the architectures of retroviral vectors and VLPs. ^{218,224 –226} Another important consideration is that CDV-based platforms that transiently deliver protein cargo are unlikely to be hampered by the same nucleic acid-induced native immune responses that restrict retroviral vectors.

For a more extensive characterization of the immune responses triggered by various components of retroviral vectors, refer to Coroadinha ²¹⁸ and Shirley et al. ²²⁴

Adding onto the landscape of innate immune responses potentially triggered by CDVs, it is relevant to mention that recent studies have revealed complex interactions between EVs and the complement system, highlighting their role in immune regulation. In addition, EVs are known to carry key complement factors as well as complement regulators that can influence inflammation. ^227,228 The exact molecular mechanisms driving these effects, and how they could impact the use of EV-based delivery vehicles for in vivo therapies, remain poorly understood and require further investigation.

Humans exhibit low preexisting immunity against retroviral vectors due to low infection rates with the wild-type viruses they are derived from. This implies that adaptive immune responses, such as production of neutralizing antibodies, are generally less likely to restrict the activity of retroviral vectors—and, by extension, of CDVs. This is in contrast with AAVs, where prior exposure and associated adaptive immunity are widespread in the human population. Adaptive responses may, however, be elicited when the retroviral vectors or CDVs are pseudotyped with envelope glycoproteins derived from viruses that more commonly infect humans, such as hemagglutinin (HA) from influenza viruses, ²²⁹ or with envelope glycoproteins derived from viruses against which vaccination is standard, such as the hemagglutinin (H) and fusion (F) peptides from the measles paramyxoviruses. ²³⁰

It is worth highlighting that, regardless of preexisting adaptive immunity, all viral fusogens are inherently immunogenic and likely to trigger innate immune responses, as well as being susceptible to inactivation via opsonization by the complement when circulating in human blood—which can undermine the safety profile and significantly hamper the efficacy of pseudotyped CDVs. Evading immune surveillance is not only paramount to ensure the safety of Cas9-CDVs but is also crucial for enhancing in vivo targeting. Modifying envelope proteins to avoid detection by neutralizing antibodies and complement, as well as to evade triggering innate immune responses, is therefore expected to significantly improve delivery efficiency. ²¹⁸ VSV-G-pseudotyped particles, for instance, are known to be inactivated by human serum. ²³¹ By contrast, alternative pseudotypes show different immune resistance profiles: LVs pseudotyped with Cocal virus envelope proteins, for example, are more resistant to the complement system, which opens possibilities for incorporating these components for in vivo applications. ²³² Another strategy is to replace viral glycoproteins with nonviral proteins with fusogenic potential. For instance, Myomaker and Myomerger are two key mammalian proteins that govern myoblast fusion and muscle formation, but are structurally and functionally divergent from traditional, viral fusogenic proteins. Hindi et al. recently pseudotyped LVs with these proteins, enabling specific delivery of therapeutic cargo to the skeletal muscle. ²³³ Similarly, the fusogenic coiled-coil peptides CPE4/CPK4, ²³⁴ the envelope protein of human endogenous retroviruses, ²³⁵ and the gamete fusion protein HAP2/GCS1 of Arabidopsis thaliana, ²³⁶ as well as human-derived CPPs, ^237,238 have been investigated for their potential to promote cell entry and cytosolic cargo release.

Importantly, a common trigger of adaptive immune responses following retroviral vector administration is the transgene they carry. Successfully transduced cells expressing a retroviral vector-encoded transgene, in fact, may be flagged as non-self by the immune system and selectively eliminated. This is due to transgene-derived peptides being presented by the major histocompatibility complex class I, which is displayed on the surface of all cells, and class II, which is exclusively expressed by professional antigen presenting cells. This triggers a signaling cascade that can produce cytotoxic T cells targeting the foreign antigen, besides promoting the synthesis of antigen-specific antibodies. ^{224,239 –241} As for other nucleic acid-related immune responses, this adaptive response is not likely to cause concern for the majority of Cas9-CDV architectures—with the exception of those that include a retroviral expression vector encoding the sgRNA or a therapeutic transgene.

Finally, viral components are not the only potential trigger of immune responses present in retroviral vectors and CDVs. For instance, HLA-I proteins, which are displayed on the plasma membrane of the CDV-producer cells, have been shown to be incorporated into the LV membrane envelope during the budding process—a mechanism also shared by CDVs. These vector-associated HLA-I proteins can elicit T cell-mediated alloimmune reactivity. As a solution, genetic ablation of the HLA-I complex in producer cells yields substantially less immunogenic LVs. ²⁴²

On top of the ubiquitous immune pathways discussed here, cell-type-specific mechanisms of antiviral defense have also been described ²¹⁸ —warranting a careful, case-by-case evaluation of the immunogenicity of CDVs redirected toward different tissues. Severe systemic immune reactions are recognized as potential complications arising from the administration of gene therapeutic products—and, in the case of high-dose AAV vectors, have sometimes proven fatal. ^243,244

LVs are the vehicle of choice for ex vivo gene therapy—more specifically, for gene addition strategies, whereas electroporation is favored for the delivery of gene editing cargo. As a consequence, data on the immunogenicity of the latest generation LVs following in vivo administration remain limited. However, the few clinical trials that have recently utilized these vectors for in vivo gene therapy have reported favorable safety profiles. ^245,246

Immune responses to the CRISPR-Cas cargo

A second concern associated with Cas9-CDVs, and common to all gene editing strategies, is the risk of immunogenicity against Cas nucleases and any derived editing tools.

Due to their bacterial origin, Cas nucleases may in fact act as pathogen-associated molecular patterns and trigger intracellular innate immune responses. Furthermore, it has been observed that—strikingly—up to 80% of individuals show preexisting humoral and cellular immunity against proteins derived from Staphylococcus aureus and Streptococcus pyogenes—including Cas nucleases. ^{247 –252} This has fueled ongoing efforts to rationally engineer minimally immunogenic nucleases for gene editing applications. ²⁵³ Anti-Cas antibodies are very likely unable to neutralize either Cas9-LVs, as they do not directly shuttle the Cas9 protein, or Cas9-CDVs, as their vesicle structure would shield the Cas9 protein cargo from immune sensing. However, the preexisting, anti-Cas immunity may lead to cytotoxic T cell responses directed against transduced cells that express Cas9, thus rendering the gene editing therapy ineffective. ^247,254 It can be hypothesized that retroviral vectors, which promote stable, long-term transgene expression, may trigger such a response more robustly than CDVs, which only transiently deliver the Cas9-sgRNA RNP.

This consideration underscores the importance of not only evaluating how the Cas9 cargo may trigger immune responses, but also how these responses may influence the success of gene editing strategies. Depending on the format of the Cas9 cargo components (e.g., retroviral expression vector or DNA encoding Cas9 and sgRNA, mRNA encoding Cas9 codelivered with chemically synthesized sgRNA, Cas9-sgRNA RNP complexes) and on the longevity of their presence in target cells, their uptake may elicit distinct cellular defense mechanisms, in turn affecting editing efficacy and cellular functions at different degrees. For example, as discussed previously, innate immune sensing mediated by activation of IFNs-1 may be triggered as a cellular protective response to retroviral infection or retroviral vector transduction. This initiates a plethora of defense mechanisms that include inhibition of reverse transcription, which can potentially impact retroviral vector-based gene transfer ²⁵⁵ but could also affect gene editing technologies that rely on the process of reverse transcription, such as prime editing. ²⁵⁶

Further investigation will be required to gain more comprehensive insights into whether delivery of Cas9-sgRNA RNP complexes via CDVs is restricted by specific cellular defense mechanisms—and, if so, how these may be circumvented.

Immune responses to the contaminants

A final concern is related to the immunogenicity risk associated with host cell contaminants present in Cas9-CDV preparations—whether they are copurified with the particles or packaged within them. The World Health Organization poses clear guidelines for the acceptable limit of residual DNA in biological products—which is fixed at 10 ng/dose, with individual fragments no longer than 200 base pairs. ²⁵⁷ To our knowledge, the residual host cell DNA encapsulated by Cas9-CDVs has never been quantified nor comprehensively characterized. However, Banskota et al. verified that less than 0.03 molecules of plasmid DNA encoding the Gag-ABE fusion protein were packaged within their Cas9-VLP^MLVs. ⁶⁹ Mangeot et al., instead, characterized the full RNA content of their Cas9-VLP^MLVs, revealing stochastic encapsulation of cellular mRNAs, rRNAs, and tRNAs, with less than 0.4% of the total RNA content being ascribable to the viral component-encoding constructs that are expressed in producer cells. ⁶⁸ A more thorough investigation of the host cell DNA copurified and packaged within Cas9-CDVs would be highly advisable, as it has been shown that a significant portion of the innate immune responses triggered following the administration of VSV-G-pseudotyped retroviral vectors are actually due to tubulovesicular structures that contaminate retroviral vector preparations and contain nucleic acids—including the plasmids used to transfect producer cells. ²⁵⁸

In contrast to DNA, there is no clear consensus on the maximum accepted content of residual host cell proteins in viral vector preparations. By the early 2000s, residual proteins derived from the producer cells had already been identified as one of the major triggers of immune reactions following LV administration, and more stringent purification methods were shown to yield markedly less immunogenic vector preparations. ²⁵⁹ However, the favorable safety profiles highlighted by recent clinical trials involving viral vectors seem to suggest that following GMPs for particle synthesis and downstream purification minimizes the risk of host cell contaminant-induced immunogenicity. ^{260 –263} Furthermore, risk-assessment studies conducted on monoclonal antibody biotherapeutics—whose purification pipelines share several similarities with those of CDVs ²⁶⁴ —encouragingly suggest low levels of host cell protein-related immunogenicity. ^265,266

The protein content of physiologically secreted EVs has been extensively characterized, and the resulting data have been compiled in publicly accessible databases such as Vesiclepedia. ²⁶⁷ However, given that the molecular profile of EVs exhibits significant variability based on vesicle type, cell type of origin, and healthy versus diseased state, it is unlikely that insights gained from these proteomic studies would be directly applicable to CDVs in general, and Cas9-CDVs in particular. This notion is further supported by the observation that distinct producer cell lines secrete EVs with unique molecular compositions, and that transfection of these cells—for example, to introduce the Cas9-encoding transgene—can alter the composition of their secretome. ^{105 –107} Mangeot et al. reported that a wide variety of host cell proteins are stochastically packaged in their Cas9-VLP^MLV preparations. The majority of these were, as expected, membrane-associated proteins and could not be detected in target cells following transduction. Conversely, cytosol-derived proteins, which were presumably stochastically packaged within the particles, could be detected in the transduced cells—although transiently. ⁶⁸

Given the novelty of CDV-based platforms for the delivery of gene editing tools, regulatory bodies will likely need to rely on the precedents established in LV regulation. Presumably, the same quality guidelines applied to LVs and other existing, similar gene therapy products may be extended to Cas9-CDVs as well. In any case, rigorous safety and efficacy assessment of Cas9-CDVs will be necessary before future clinical trials.

In conclusion, the regulatory landscape for CDVs harboring CRISPR tools is rapidly evolving, and we can anticipate that the guidelines for their manufacturing and downstream processing will be updated to account for the complexity of CDVs as therapeutic products—following the example set by AAVs produced as vectors for gene therapy. ²⁶⁸ Indeed, the field is hampered by the limitations of current analytical techniques, which are still not capable of providing detailed and in-depth characterization of all product-associated impurities. To bridge this gap, the European Medical Agency and the U.S. Food and Drug Administration published guidelines that promote the adoption of risk-based approaches balancing purity concerns with demonstrated in vivo safety and efficacy. ²⁶⁹ Moving forward, further studies, continuous monitoring of data from ongoing clinical trials, and development of more sensitive analytical techniques will be paramount to establish specific quality parameters and ensure robust safety profiles.