The clinical progress of genome editing technology and their in vivo delivery techniques

Abstract

There is wide interest in applying genome editing tools to prevent, treat, and cure a variety of diseases. Since the discovery of the CRISPR/Cas9 systems, these techniques have been used in combination with different delivery systems in order to create the treatment option with high efficacy. Each delivery system has its own advantages and disadvantages and is being used for various applications. With a large number of gene editing applications being studied but very few being brought into the clinic, there is a need to review the current progress in the field, specifically where genome editing has been applied in vivo and, in the clinic, and identify areas of future growth and current challenges.

1. The fundamentals of CRISPR/Cas9 genome editing technologies

Since 2013 with the discovery that clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) can effectively edit the eukaryotic genome, there has been a rush to perfect the delivery mechanism [1–5]. CRISPR comes on the back of multiple genetic innovations such as designer zinc finger nucleases (ZNFs) and transcription activator-like effectors (TALENs) [6, 7]. However, the bulk of genome editing is now conducted via CRISPR. There are a variety of different methods for delivery of CRISPR/Cas9 including viral vectors, lipid nanoparticles (LNPs), polymers, and gold nanoparticles (AuNPs). The most promising of these therapies have already begun clinical trials for a variety of different indications and although these trials are slow moving, there is likely to soon be a huge amount of innovation for both in vivo and ex vivo treatments using CRISPR technologies [8]. There are relatively few clinical trials, when compared to the surplus of academic literature, that within the next five years it is expected for there to be a large uptick of trials in the clinical setting.

Genome editing is typically directed by the Streptococcus pyogenes (S. pyogenes) type II CRISPR/Cas nuclease Cas9 and a chimeric single guide RNA (sgRNA), which is formed by the fusion of CRISPR RNA (crRNA) with a trans-activating crispr RNA (tracrRNA) [1, 2, 9, 10]. crRNA contains a 20-nucleotide sequence homologous to a target DNA site which is known as a protospacer [9]. Once the formation of the Cas9/sgRNA ribonucleoprotein complex is complete, Cas9 is led to the sequence specific protospacer and, in the presence of downstream trinucleotide 5’-NGG-3’ protospacer adjacent motif (PAM), the DNA is cleaved in the middle of positions 17 and 18 of the protospacer. In mammalian cell lines, double strand break formations enlist endogenous DNA repair pathways which can result in multiple different editing outcomes. Non-homologous end joining (NHEJ) serves as the major repair pathway, where blunt DNA ends are fixed in an error-prone fashion, yielding many small insertions or deletions (indels)[11]. A different pathway to produce a precise genome modification exploits the homology-directed repair (HDR) pathway. This uses an exogenous donor DNA template that contains the desired edit and is co-introduced with Cas9 and sgRNA. Additionally, point mutations in one or both of the catalytic RuvC and HNH domains of Cas9 has enabled the improvement of catalytically disabled Cas9 variants that either nick a single DNA strand (Cas9 nickase, nCas9) or are inactive (dead Cas9, dCas9). The classical use of NHEJ and HDR editing can be greatly expanded by the use of these variants [12, 13]. Many of these new editors have been developed to offer alternative editing techniques and overcome some of the challenges of Cas9. One new editing technique that utilizes nCas9 fused to reverse transcriptase (RT) is prime editing (PE) [14–16]. Prime editing uses an introduced prime editing guide RNA (pegRNA) that guides the system to the desired DNA site and encodes the specific edit to replace the original DNA, allowing very specific base edits without changes to untargeted bases in the activity window [14–16]. Another new editing technique that utilizes nCas9 is base editing (BE), which fuses nCas9 and a deaminase enzyme to induce single base substitutions at a target site [14, 17–20]. dCas9 is used in another new editing technique known as epigenome editing which modulates gene expression and directs cell differentiation [21, 22]. Alternative Cas proteins such as Cas12 and Cas13 are derived from alternative organisms and offer unique differences compared to Cas9 that pose additional advantages to the genome editing tool. One of the most popular Cas12 enzymes is known as Cas12a (or formerly known as Cpf1). One of the major differences between it and Cas9 is that it does not need tracrRNA and allows for the processing of its own guide RNA [23, 24]. Cas12a is commonly used for multiplex genome editing [24]. On the other hand, Cas13 is another commonly used alternative Cas protein and is unique because it targets RNA instead of DNA, which makes it beneficial for altering gene expression without affecting the genome sequence [25].

2. In vivo delivery systems of CRISPR/Cas9 for therapeutic applications

Successful genome and epigenome editing always includes efficient delivery of CRISPR/Cas9 reagents into mammalian cell nuclei. Reagents are delivered as either DNA, RNA, or pre-assembled ribonucleoproteins (RNPs) which are complexes between Cas9 protein and sgRNA [17, 26]. The use of RNPs for genome editing in various applications is further reviewed by Bloomer et al 2021 [17]. However, successful and efficient delivery methods are necessary for clinical use and development of these genome editing techniques. CRISPR/Cas9 has been delivered into different target cells and organs in the body via multiple delivery systems. These systems have evolved over the years to increase efficiency and specificity and lower toxicity. The most popular delivery systems include viral vectors, lipid nanoparticles, polymers, and inorganic nanoparticles (Figure 1). A summary of these delivery systems is shown in Table 1.

Figure 1.

The four main delivery systems on CRISPR/Cas9. Viral vectors, lipid nanoparticles (LNPs), polymers, and inorganic nanoparticles.

Table 1.

Summary of most popular genome editing delivery systems.

Delivery System	Advantages	Disadvantages	Stage of Development	References
Viral Vectors	-easy small-scale production High transduction efficiency -organ/cell specificity	-high off target risk -potential integration into host genome -high cost	Clinical	[24,28–32]
Lipid Nanoparticles	-low toxicity -high biocompatibility -biodegradable -organ/cell specificity	-long term instability	Clinical	[23,32,39,43]
Polymers	-sustained and controlled release -large payload capacity	-long degradation time	Preclinical	[45,47]
Inorganic Nanoparticles	-tunable composition, morphology, and photoelectrochemical properties	-limited drug loaded -accumulation in organs	Preclinical	[48–54]

2.1. Viral vectors

Viral vectors are some of the most widely used delivery vehicles for genome editing tools mainly due to their easy manufacturing and high transduction efficiency [27]. Some of the most commonly used viral vectors for genome delivery are adeno associated virus (AAV), adenovirus (AV), and lentivirus (LV).

AAVs are popular for in vivo studies because they have a low toxicity due to limited off target effects. One study used CRISPR/Cas9 delivered with AAVs for treatment of Duchenne Muscular Dystrophy (DMD) in mice and found that the treatment requires high doses of AAV, up to 1.8 × 10¹⁵ vector genomes (vg)/kg, due to the skeletal tissue being such a large part (~40%) of the body. The issue with such a large dose is that it may lead to acute liver toxicity. In this study, the researchers aimed to reduce the necessary amount of AAV required when attempting a deletion of the Dmd exon 44 gene by co-delivering single stranded AAVs (ssAAVs) with Streptococcus pyogenes (Sp) Cas9 and self-complementary AAVs (scAAVs) with sgRNAs with varying doses of ssAAVs packaged sgRNAs and achieved 40%, 32%, 95% and 95% dystrophin-positive myofibrils in the tibialis anterior, triceps, diaphragm, and heart, respectively [28].

AVs have additionally been used as delivery systems for genome editing tools. AVs pose several advantages as delivery systems due to their high transduction efficiency, however they often induce high immunogenicity. In one study, researchers used AVs to deliver Base Editor 3 (BE3) to the Angptl3 gene to cause a loss of function mutation in mice for reduction of blood lipid levels [29]. This study showed a decrease in blood levels of triglycerides (56%) and cholesterol (51%) 14 days after intravenous injection [29].

LVs have been used in various applications including the delivery of Cas9 mRNA and vascular endothelial growth factor A (Vegfa) gRNA in mice to prevent wet age-related macular degeneration [30]. Researchers of this study were able to knock out 44% of Vegfa in the retinal pigment epithelium and reduce choroidal neovascularization by 63% [30].

Although viral vectors have been widely studied, they are currently limited for in vivo applications due to their associated toxicity [31, 32]. Additionally, the delivery of viral vectors or plasmids that encode Cas9 and sgRNA can possibly lead to integration into the host genome [33]. If these components are integrated into the genome randomly, then insertional mutagenesis at critical genomic sites may occur, leading to gene disruption or oncogenesis. Repeated expression of Cas9 cassettes increases the chance of off-target Cas9 editing activity [34]. Furthermore, the introduction of foreign bodies can cause a host immune response thus limiting the efficacy for nucleic acid approaches therapeutically [35].

2.2. Lipid Nanoparticles (LNPs)

Lipid nanoparticles (LNPs) are commonly used to deliver CRISPR/Cas9 into cells due to their low toxicity, high biocompatibility, and prolonged circulation time [26]. LNPs typically are formulated with various amine head and tails that aid in the specific cell/organ targeting and with helper lipids and excipients that help with their overall delivery efficiency [36, 37]. For example, in mouse primary T lymphocytes, various combinations of amine heads and tails were screened to see their targeting ability and efficiency and the researchers achieved 8.2% gene recombination from intravenous injections of LNPs containing Cre mRNA with one of their anime combinations [38]. One of the most common helper lipids is polyethylene glycol (PEG)-ylated lipid which contributes to the “stealth” properties of LNPs, allowing for an increase in the time they spend in circulation before being cleared [39]. Additional clinical advantages of LNPs are that they can be produced in large batches and easily modified for specific applications [40, 41]. The mechanism of entry of LNPs into cells is endocytosis, which allows for the LNPs to release their cargo into the cytoplasm of the cells [33]. LNPs have been used extensively to effectively deliver to specific cells and organs in vivo including but not limited to the liver, brain, muscle, and lungs [35, 42]. For example, the Angiopoietin- like 3 (Angptl3) gene in the liver was edited as a treatment for human lipoprotein metabolism disorders by delivering Cas9 mRNA in LNPs via intravenous injections [42]. The researchers achieved genome editing of 38.5% and a 65.2% serum reduction of the ANGPTL3 protein in mice [42].

LNPs have also been used in delivering ribonucleoproteins (RNPs) for in vivo genome editing. For example, RNPs were delivered into mice for editing in the cochlea using a commercially available LNP, Lipofectamine 2000, and delivered a base editor RNP (BE3 RNP), which induced a C to T conversion at a specific target site with limited off target effects [43]. In the retina, the vascular endothelial growth factor A (Vegfa) knockout was attempted by delivering LNPs containing RNPs into the subretinal space in mice and achieved 6% indels in Vegfa of isolated EGFP-positive RPE cells [44].

Additionally, in the preclinical setting, LNPs have become a prominent delivery mechanism. For example, VERVE-101, a therapeutic developed by Verve Therapeutics, treats Heterozygous Familial Hypercholesterolemia[45]. This therapy is a liver targeting LNP loaded with a CRISPR base editor that knockdowns the PCSK9 gene and causes reduction in serum levels of low-density lipoprotein cholesterol [45]. This therapy particularly shows promise for future clinical trials. The first clinical trial is scheduled to begin by the middle of 2022 and has already been approved for phase 1.

Although LNPs have many advantages that make them a promising delivery system for delivering genome editing techniques for various clinical applications, they also pose some challenges that need to be addressed for the further development in the clinic including their long-term stability and specificity. As LNPs are stored over time, aggregation may occur [46]. The aggregation of LNPs may result in a change in their shape, size, surface area, and surface charge leading to an altered in vivo distribution and efficacy in the body.

2.3. Polymers

There are a variety of polymers that are being used as delivery systems for genome editing tools. Polymers are most commonly formulated into nanoparticles, micelles, and dendrimers [47]. Some of the most common polymers being used are poly(lactic-co-glycolic acid) (PLGA), poly(ethylenimine) (PEI), poly(amido amine) (PAMAM), and chitosan. Some of the advantages of using polymers include their sustained release and ability to carry large cargo sizes [48]. One recent study used chitosan nanoparticles to co-deliver Doxorubicin (DOX) and Survivin CRISPR/Cas9 expressing plasmid (sgSurvivin pDNA) to cancer cells in mice in order to downregulate expression of Survivin by knocking out the gene expression and resultantly enhance the DOX activity[49]. Researchers of this study achieved 42.0% apoptosis with NPs containing DOX and sgSurvivin pDNA [49]. Polymers also pose a unique property that allows for a controlled release; they may release their cargo in response to external triggers such as light, temperature, pH, and pressure [47]. Although polymers may be advantageous for delivering genome editing tools in vivo, they also pose challenges. Polymers, especially cationic ones, have shown to exhibit high toxicity in vivo, which can resultantly cause an unwanted immune response [50].

2.4. Inorganic Nanoparticles

Inorganic nanoparticles have been commonly used as delivery systems for genome editing tools due to their general low toxicity and ability to deliver donor DNA [51–53]. The most commonly used inorganic nanoparticles include gold nanoparticles (AuNPs), mesoporous silica nanoparticles (MSNPs), and metal organic frameworks (MOFs).

AuNPs have been used as a delivery method for different disease indications including treatment of fragile X syndrome (FXS) in mice [52]. In this study, CRISPR/Cas9 and Cpf1 were delivered using AuNPs to target the metabotropic glutamate receptor 5 (mGLuR5) via intracranial injection in mice and achieved an editing frequency of 14.6% with no off-target effects [52]. Although potentially promising, AuNPs pose concerns associated with their accumulation in the liver and spleen and aggregation [54].

Additionally, MSNPs have proven to be promising delivery systems due to their easy surface manipulation, biocompatibility, biodegradability, and low cost [55]. MOFs have become increasingly popular as delivery systems for genome editing tools due to their adjustable pore size and ability to protect their cargo [56, 57]. One major drawback of this approach may be the limited drug loading depending on the specific procedure used [50].

3. Clinical trials of CRISPR therapeutics

Once therapeutics have been thoroughly researched and optimized, they may enter into clinical trials where the goal is to gain FDA approval and make it to the market. However, there is a large gap between the number of therapies being researched compared to the amount that are actually in clinical trials. The delivery systems can be broken into two different categories: in vivo and ex vivo. A summary of the active clinical trials is provided in Table 2.

Table 2.

Comprehensive List of current genone editing clinical trials

ClinicalTrails.gov Identifier	Intervention/Treatment	Mechanism	Ex/In vivo	Condition or Disease	Phase	Status	Year
NCT03872479	EDIT-101	CRISPR-Cas9-mediated removal of CEP290 mutation delivered by AAV, subretinal injection	In vivo	Leber Congenital Amaurosis 10-IVS26	I/II	Recruiting	2019
NCT04560790	BD111	CRISPR-Cas9 mRNA-mediated HSV-1 genome disruption, corneal injection	In vivo	Refractory Herpetic Viral Keratitis	I/II	Active, not recruiting	2020
NCT04601051	NTLA-2001	CRISPR-Cas9-mediated TTR knockout delivered by LNP, intravenous injection	In vivo	Hereditary Transthyretin Amyloidosis	I	Recruiting	2020
NCT05120830	NTLA-2002	CRISPR-Cas9-mediated KLKB1 knockout delivered by LNP, intravenous injection	In vivo	Hereditary Angioedema	I/II	Recruiting	2021
NCT03398967	Universal Dual Specificity CD19 and CD20 or CD22 CAR-T Cells	Universal CRISPR-Cas9 gene-edited allogeneic CAR-T cells targeting CD19 and CD20 or CD22	Ex vivo	Relapsed or Refractory Leukemia and Lymphoma	I/II	Unknown	2018
NCT04990557	PD-1 and ACE2 Knockout T Cells	CRISPR-Cas9-mediated knockout of PD-1 and ACE2 in T cells to induce long-term immunity for COVID-19	Ex vivo	COVID-19 Respiratory Infection	I/II	Not yet recruiting	2021
NCT04774536	CRISPR_SCD001	CRISPR-Cas9-mediated HBB correction in CD34+ HSPCs	Ex vivo	Severe Sickle Cell Disease	I/II	Not yet recruiting	2021
NCT04035434	CTX110	CRISPR-Cas9 gene-edited allogeneic CAR-T cells targeting CD19	Ex vivo	Relapsed or Refractory B-Cell Malignancies	I	Recruiting	2019
NCT05066165	NTLA-5001	CRISPR-Cas9 gene-edited autologous TCR-T cells targeting WT-1	Ex vivo	Acute Myeloid Leukemia	I	Recruiting	2021
NCT04037566	Genetic: XYF19 CAR-T cells Drug: Cyclophosphamide Drug: Fludarabine	CRISPR-Cas9 gene-edited autologous CD19 CAR-T cells with HPK1 disruption	Ex vivo	Relapsed or Refractory CD19+ Leukemia or Lymphoma	I	Recruiting	2019
NCT04426669	Biological: Tumor-Lymphocytes (TIL) Drug: Cyclophosphamide Drug: Fludarabine Drug: Aldesleukin	CRISPR-Cas9-mediated CISH lymphocytes	Ex	Metastatic Epithelial Cancer	I/II	Recruiting	2020
NCT04925206	ET-01	CRISPR-Cas9-mediated BCL11A disruption in CD34+ HSPCs	Ex vivo	β-Thalassemia	I	Active, not recruiting	2021
NCT04637763	Genetic: CB-010 Drug: Cyclophosphamide Drug: Fludarabine	CRISPR-Cas9 gene-edited allogeneic CAR-T cells targeting CD19	Ex vivo	Relapsed or Refractory B Cell Non-Hodgkin Lymphoma	I	Recruiting	2020
NCT03545815	Anti-mesothelin CAR-T cells	CRISPR-Cas9 gene-edited PD-1 and TCR knockout mesothelin-directed CAR-T cells	Ex vivo	Mesothelin Positive Multiple Solid Tumors	I	Recruiting	2018
NCT04557436	PBLTT52CAR19	CRISPR-Cas9 edited CD52 and TRAC CAR-T cells targeting CD19	Ex vivo	B-cell Acute Lymphoblastic Leukemia	I	Recruiting	2020
NCT04819841	GPH101	CRISPR-Cas9-mediated HBB correction in autologous CD34+ HSPCs	Ex vivo	Sickle Cell Disease	I/II	Recruiting	2021
NCT05329649	CTX001	CRISPR-Cas9-mediated BCL11A disruption in autologous CD34+ HSPCs	Ex vivo	Severe Sickle Cell Disease	III	Recruiting	2022

3.1. In vivo deliveries

There are over fifty active studies pursuing CRISPR/Cas9 as the main form of intervention for a variety of indications, however only a very small portion of those are applied in vivo. For clinical trial enrollment, recruitment is typically slow and has been further delayed due to the COVID-19 Pandemic. Many of the indications targeted are typically considered orphan designations.

There are a few other clinical trials that look to treat a variety of indications. These indications range from single point mutation causing illnesses to complex polygenic traits.

One of these trials uses a subretinal injection to deliver AAV carrying CRISPR-Cas9 to remove CEP290 for Leber Congenital Amaurosis 10-IVS26 (ClinicalTrials.gov ID: NCT03872479). The treatment of interest, EDIT-101, is a novel gene editing product designed to eliminate the mutation on the CEP290 gene. Additional goals of this study are to measure the frequency of adverse events, determine the number of participants experiencing procedural related adverse events, and calculate dose limiting toxicities. Similar to many other CRISPR based trials there is difficulty in recruiting patients for this trial particularly because the trial focuses on pediatric patients.

Additionally, Intellia Therapeutics has two clinical developments, both currently in phase 1, that are showing great potential for all in vivo clinical results: NTLA-2001 and NTLA-2002. The trial (NCT04601051) which uses NTLA-2001 consists of an open-label, single ascending dose which may identify the optimal biologically active dose (OBD) of NTLA-2001, followed by Part 2, if applicable, an open-label, single- dose expansion at the OBD to further characterize activity of NTLA-2001, provide an initial assessment of the effect of NTLA-2001 on clinical measures of neuropathy and neurologic function, and obtain additional safety data at the OBD. The trial’s (NCT05120830) purpose is to evaluate the safety, tolerability, activity, pharmacokinetics, and pharmacodynamics of NTLA-2002 in adults with Hereditary Angioedema. Overall, there are limited amounts of in vivo clinical trials due to complications in the delivery system, the efficacy of in vivo delivery, and regulatory complications of gene editing the human genome. As these issues slowly get resolved, there will be significantly more in vivo clinical trials.

3.2. Ex vivo systems

In addition to in vivo systems, ex vivo systems are being studied for a large range of indications and vary greatly in their trial design. The majority of CRISPR clinical trials are the ex vivo systems. Some of the most promising and noteworthy clinical trials are detailed in this paragraph. A lot of these ex vivo systems utilize CAR-T cells for genome editing. One particular study worth highlighting includes one of the most effective uses of gene therapy while simultaneously highlighting the issues with recruitment, Anti-mesothelin CAR-T cells for treatment of Mesothelin Positive Multiple Solid Tumors (ClinicalTrials.gov ID: NCT03545815). This study’s purpose is to evaluate the safety and efficacy of CRISPR-Cas9 mediated PD-1 and TCR gene knockout. This study also looks to analyze the persistence of transferred CAR-T cells and observe and measure anti-tumor responses for patients with detectable mesothelin positive tumor lesions. However, there have been many problems recruiting participants, which is a common problem with many other CRISPR based clinical trials. The study began on March 19, 2018, and was expected to be completed by late 2020, however, the trial is still ongoing.

Ex vivo systems are leading the clinical trials compared to in vivo systems but still are sparse in comparison to the number of applications currently being researched in the field trying to get to the clinical trial point.

4. Concluding Remarks and Future Perspectives

The CRISPR/Cas9 system has been widely studied in academic context however, in terms of clinical application there has been little overall development. Many current CRISPR/Cas9 delivery mechanisms have issues with either safety or efficacy that make their in vivo application difficult. It seems that there is still a great deal of work to be done to translate the research being done in academia into the clinic. On the other hand, however, there are many ex vivo CRISPR/Cas9 applications that have made their way through clinical trials and are already on the market. For deeper insight into some of the questions still remaining in this field see Outstanding Questions.

Outstanding Questions.

• When will the lag between academic papers and clinical trials significantly lessen?

• Will viral vectors remain the dominant form of delivery or will another type of mechanism be proven safer and more effective?

• Will clinicians and patients readily accept gene editing technologies even if they are proven to be effective?

• Can the CRISPR-Cas9 system produce more efficient gene editing?

• What are the potential long term downstream effects for extended use of CRISPR for in vivo subjects?

• Will the CRISPR-Cas9 system be able to effectively target different organs as effectively as it targets the liver?

• The CRISPR-Cas9 system has been around in its current form since 2013. What will the next big innovation be in the gene therapy space?

• What is the short- and long-term toxicity of each modality as well as the effects of CRISPR itself?

• What is the relationship between CRISPR-Cas activity and horizontal gene transfer (HGT)?

• Will there be additional regulation of CRISPR when compared to traditional treatments? Will that regulation be different for children?

• What indications are going to be easiest for CRISPR to cause an impact right away?

• Will genome editing ever be implemented in germ cells?

Genome editing tools have come a long way since their initial development and use with ZFNs and TALENs and have continued to evolve over the past decade since the discovery and resultant implementation of CRISPR/Cas systems in 2013. As CRISPR systems continue to evolve into more specific mechanisms such as base and epigenome editing, there will likely be the rise of more efficient and safe tools. CRISPR has continued to break boundaries and with the incredible amount of academic research and publications, imminently there will be available treatments for patients. Additionally, CRISPR technology can be used in multiple different species for the potential cure of human diseases like malaria. In the future, CRISPR could be used for a variety of different indications spanning from cancer to obesity in addition to more precarious ethical questions such as the development of “Designer babies” by controlling specific human attributes such as physical appearance and athletic ability.

Currently, viral vectors are the dominant delivery system for genome editing tools in vivo as they have been the most studied thus far due to their ease of production and high efficiency. They have also been widely used for ex vivo approaches, as they have shown to have high delivery efficacy. However, there are other systems that have shown more promise than viral vectors, such as lipid nanoparticles, polymers, and gold nanoparticles. These other systems are the subject of a tremendous amount of research and will continue to be improved until they are more effective as the existing types of viral vectors. In particular, LNPs show great potential for delivering CRISPR/Cas9 into target cells and organs due to their sustained circulation time, low toxicity, and high targeting specificity. These LNPs, as well as some other systems, have been able to successfully target most commonly the liver, but also different organs such as the lungs and spleen [38, 58, 59]. LNPs can also be produced very cheaply at a large scale. However, it is imperative for the future of medicine to increase the targets to other cells and organs to expand into other disease implications. The brain is of particular interest for targeted drug delivery since there are a limited number of drugs that can effectively cross the blood brain barrier (BBB) and the development of delivery systems that can carry various cargoes across the BBB could greatly expand the potential applications for disease treatment in this region. Besides LNPs, polymers and AuNPs have also shown promise as a potential delivery mechanism however each of these have their own associated drawbacks. Polymers are particularly useful to carry large payloads and can be activated by external stimuli but there is little data on long term toxicity. AuNPs are known for their low toxicity and ability to deliver DNA however there are potential problems with aggregation and unknown long-term toxicity as they tend to accumulate in the liver and spleen. The development of more refined targeted delivery systems is essential not only for increasing efficacy but also for reducing off-target effects and thus reducing the toxicity and safety issues associated with them. The greater the delivery system is at achieving only on target editing, the less safety concerns arise. The most crucial development of in vivo genome editing tools, most specifically CRISPR/Cas9, is to continue to optimize their delivery systems in order to use these tools outside of the laboratory setting. Future clinical trials are reliant on the development of these delivery systems, and as they continue to improve, we may likely see CRISPR/Cas9 trials and their resultant market availability truly open in the clinical setting as we have seen in the academic context.

Highlights.

• Genome editing poses great potential for in vivo applications but still must overcome some challenges

• There are many modes or delivery of genome editing tools including viral vectors, lipid nanoparticles, polymers, and inorganic nanoparticles

• There is a large gap between the amount of genome editing therapies in academia compared to the amount in clinical trials.

• Viral vectors are currently predominantly used in vivo the current gold standard, but lipid nanoparticles can be used to improve on them