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<h2>Introduction</h2> <p><a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/malignant-neoplasm" title="Learn more about Malignant Neoplasm">Cancer</a>&nbsp;is among the foremost medical problems in the developed world today. In Canada alone, 2 out of 5 people will develop cancer in their&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lifespan" title="Learn more about Lifespan">lifetime</a>, and 1 in 4 people will die of cancer&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0005" name="bbib0005">[1]</a>. Cancerous cells evolve rapidly and are heterogeneous, and are therefore extremely difficult to target specifically. The disease is traditionally treated using [a combination of]&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/chemotherapy" title="Learn more about Chemotherapy">chemotherapy</a>,&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/radiation" title="Learn more about Radiation">radiation</a>, and surgery. Although therapies involving cytotoxic&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/chemical-agent" title="Learn more about Chemical Agent">chemical agents</a>&nbsp;and radiation are sometimes effective, they can have mixed outcomes and cause severe health&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/side-effect" title="Learn more about Side Effect">side effects</a>&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0010" name="bbib0010">[2]</a>. Meanwhile, surgery is only effective in early cases where the cancer remains confined. As our understanding of cancer has progressed, research has shifted towards elucidating the molecular&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/base" title="Learn more about Base">basis</a>&nbsp;of cancer for the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/development" title="Learn more about Development">development</a>&nbsp;of targeted cancer treatments. This is reflected in the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/prevalence" title="Learn more about Prevalence">prevalence</a>&nbsp;of&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/drug" title="Learn more about Drug">drugs</a>&nbsp;which produce specific molecular alterations&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0010" name="bbib0010">[2]</a>. However, targeted therapies are also possible at the genetic level, forming the basis for the application of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/nested-gene" title="Learn more about Nested Gene">gene</a>&nbsp;therapy to treat cancer.</p> <p>Gene therapy research has only emerged in the past two decades. The aim of this relatively young field is to edit and deliver&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/recombinant-dna" title="Learn more about Recombinant DNA">recombinant DNA</a>&nbsp;for therapeutic purposes. Gene therapies are promising treatments for many diseases because they can be both&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/chlorophacinone" title="Learn more about Chlorophacinone">quick</a>&nbsp;to develop and specific at the molecular level. However, there have been setbacks at the clinical stage. During a high-profile 1999 gene therapy trial for&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/ornithine" title="Learn more about Ornithine">ornithine</a>&nbsp;transcarbamoylase (OTC) deficiency, participant Jesse Gelsinger died of an&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immune-response" title="Learn more about Immune Response">immune response</a>&nbsp;to the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/adenovirus-vector" title="Learn more about Adenovirus Vector">adenoviral vector</a>&nbsp;used for delivering the corrective gene&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0015" name="bbib0015">[3]</a>. Almost at the same time, a highly-publicized gene therapy trial to treat X-linked severe combined deficiency (X-SCID) with&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/retrovirus" title="Learn more about Retrovirus">retrovirus</a>&nbsp;was completed. The therapy seemed like a huge success at first, but some patients later developed cancer due to vector integration with nearby&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/proto-oncogene" title="Learn more about Proto Oncogene">proto-oncogenes</a>, among other genetic abnormalities&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0020" name="bbib0020">[4]</a>. Interest and confidence in gene therapy suffered as a result.</p> <p>However, the field is making a comeback as recent clinical trials for various diseases are producing promising results. Currently cancer is the major target of gene therapy, making up about 65% of gene therapy clinical trials as of 2012&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0025" name="bbib0025">[5]</a>. ​In 2016, out of 66 gene therapy clinical trials compiled by the Journal of Gene Medicine, 46 trials were targeted towards some form of cancer therapy&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0030" name="bbib0030">[6]</a>. Although our knowledge of&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/genetic-disorder" title="Learn more about Genetic Disorder">genetic diseases</a>&nbsp;such as OTC deficiency and&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/x-linked-severe-combined-immunodeficiency" title="Learn more about X Linked Severe Combined Immunodeficiency">X-SCID</a>&nbsp;is extensive, efficient and safe transfer of recombinant DNA products has been the main setback in the past. Thus the key factor contributing to successes today are improvements in gene therapy delivery .</p> <h2>CRISPR-Cas9 technology</h2> <p>In the past five years&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genome-editing" title="Learn more about Genome Editing">genome editing</a>&nbsp;technologies using clustered regularly interspersed palindromic repeats (CRISPR) in combination with CRISPR-associated systems (Cas) have revolutionized the field&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0035" name="bbib0035">[7]</a>. This technology has become so versatile and accessible that it has been adopted in academic labs worldwide and has been featured in more than 5000 publications on&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/medline" title="Learn more about Medline">PubMed</a>since 2013. The deployment of CRISPR-Cas technologies has even given&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/clotiazepam" title="Learn more about Clotiazepam">rise</a>to&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/human" title="Learn more about Human">human</a>&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/germline" title="Learn more about Germline">germline</a>&nbsp;editing discussions&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0035" name="bbib0035">[7]</a>. As of 2016, two trials for&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/malignant-neoplasm" title="Learn more about Malignant Neoplasm">cancer</a>treatment have been announced in China and the United States, both of which will utilize CRISPR-Cas to engineer patient&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/t-cells" title="Learn more about T Cells">T cells</a>&nbsp;<em>in vitro</em>&nbsp;to destroy cancer cells&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0040" name="bbib0040">[8]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0045" name="bbib0045">[9]</a>. Although gene therapy makes up less than 5% of interventional cancer studies worldwide at present, this&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/development" title="Learn more about Development">development</a>&nbsp;has considerable promise<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0050" name="bbib0050">[10]</a>.</p> <p>Implementation of this technology requires a Cas&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/nuclease" title="Learn more about Nuclease">nuclease</a>, such as the widely-used nuclease from the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacterium" title="Learn more about Bacterium">bacterium</a>&nbsp;<em><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/streptococcus-pyogenes" title="Learn more about Streptococcus pyogenes">Streptococcus pyogenes</a></em>&nbsp;(Cas9), to be expressed within the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/target-cell" title="Learn more about Target Cell">target cell</a>. To function,&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cas9" title="Learn more about Cas9">Cas9</a>&nbsp;requires a&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/guide-rna" title="Learn more about Guide RNA">guide RNA</a>&nbsp;(gRNA) composed of a scaffold sequence for Cas9 binding and a spacer sequence that defines the target region&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0055" name="bbib0055">[11]</a>. Injecting naked&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/plasmid" title="Learn more about Plasmid">plasmids</a>&nbsp;encoding Cas9 and a gRNA into the bloodstream results in very low levels of gene editing in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mouse" title="Learn more about Mouse">mice</a>&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0060" name="bbib0060">[12]</a>. New delivery methods for CRISPR-Cas9 must be efficient, non-toxic, evade immune&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/clearance" title="Learn more about Clearance">clearance</a>, and in the case of cancer therapy, deliver specifically to&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/neoplasm" title="Learn more about Neoplasm">tumor</a>&nbsp;cells. Here we review the&nbsp;<em>in vitro</em>&nbsp;and pre-clinical advances in developing viral and&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/non-viral-vector" title="Learn more about Non-Viral Vector">non-viral vectors</a>&nbsp;for CRISPR-Cas9 delivery made within the past few years, and discuss their implications for cancer gene therapy (<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#fig0005" name="bfig0005">Fig. 1</a>).</p> <p><img alt="Figure 1" src="https://ars.els-cdn.com/content/image/1-s2.0-S1740675717300026-gr1.jpg" style="height:554px" /></p> <ol> <li><a href="https://ars.els-cdn.com/content/image/1-s2.0-S1740675717300026-gr1_lrg.jpg" target="_blank" title="Download high-res image (691KB)">Download high-res image (691KB)</a></li> <li><a href="https://ars.els-cdn.com/content/image/1-s2.0-S1740675717300026-gr1.jpg" target="_blank" title="Download full-size image">Download full-size image</a></li> </ol> <p>Figure 1.&nbsp;Local and systemic delivery of CRISPR-Cas9 to target&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/malignant-neoplasm" title="Learn more about Malignant Neoplasm">cancer</a>&nbsp;cells. (a)&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/virus-vector" title="Learn more about Virus Vector">Viral vectors</a>&nbsp;and synthetic&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/nanoparticle" title="Learn more about Nanoparticle">nanoparticles</a>&nbsp;can be injected intravenously, for&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/systemic-administration" title="Learn more about Systemic Administration">systemic administration</a>; or intratumorally, for direct delivery. Vectors delivered systemically must travel through the endothelial wall and through&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/neoplasm" title="Learn more about Neoplasm">tumor</a>&nbsp;tissues before transducing or transfecting cancer cells. Alternatively, hydrogels can be used for sustained, local delivery of nanoparticles near the tumor site. (b)&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/adenovirus-vector" title="Learn more about Adenovirus Vector">Adenoviral vectors</a>&nbsp;deliver&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/dna" title="Learn more about DNA">DNA</a>&nbsp;coding for&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cas9" title="Learn more about Cas9">Cas9</a>and gRNA into the cells by binding to specific&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/molecule" title="Learn more about Molecule">molecules</a>&nbsp;on the cell&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/surface-property" title="Learn more about Surface Property">surface</a>, triggering&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/endocytosis" title="Learn more about Endocytosis">endocytosis</a>. Synthetic nanoparticles can deliver Cas9 in&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/protein" title="Learn more about Protein">protein</a>&nbsp;form, complexed with gRNA. Nanoparticles are taken up by endocytosis, fuse with the endosomal membrane, and&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/escape-behavior" title="Learn more about Escape Behavior">escape</a>&nbsp;into the cytosol, releasing their payload into the cells.</p> <h2>Viral vectors</h2> <p>By far the most popular tools for delivery of CRISPR-Cas9-mediated gene therapy today are&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/virus-vector" title="Learn more about Virus Vector">viral vectors</a>, which made up 66% of gene therapy trials as of 2012&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0065" name="bbib0065">[13]</a>. The biggest challenge of employing viral vectors is to&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/ensure" title="Learn more about Ensure">ensure</a>&nbsp;that they are specific in their target and in their&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/tropism" title="Learn more about Tropism">tropism</a>&nbsp;(i.e., affinity for a select cell type)&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0070" name="bbib0070">[14]</a>. To compound the specificity problem, CRISPR-Cas9&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genome-editing" title="Learn more about Genome Editing">gene editing</a>&nbsp;is also subject to off-target effects which have been extensively analyzed&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0075" name="bbib0075">[15]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0080" name="bbib0080">[16]</a>.</p> <p>Adeno-associated&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/virus" title="Learn more about Virus">viruses</a>&nbsp;(AAVs),&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/adenovirus-vector" title="Learn more about Adenovirus Vector">adenoviral vectors</a>&nbsp;(AdVs), and&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/retrovirus" title="Learn more about Retrovirus">retroviruses</a>have comprised the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/volume" title="Learn more about Volume">bulk</a>&nbsp;of gene therapy trials to date. AdVs hold the advantage of carrying larger constructs than AAVs, with a higher level of associated&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/protein" title="Learn more about Protein">protein</a>expression, and have been employed to carry&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cas9" title="Learn more about Cas9">Cas9</a>&nbsp;with resulting targeting efficiency comparable to rates achieved with&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/transcription-activator-like-effector-nuclease" title="Learn more about Transcription Activator-Like Effector Nuclease">transcription activator-like effector nucleases</a>&nbsp;(TALENs)&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0085" name="bbib0085">[17]</a>. Retroviruses convert their&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/genomic-rna" title="Learn more about Genomic RNA">viral RNA genome</a>&nbsp;to&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/dna" title="Learn more about DNA">DNA</a>via&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/reverse-transcriptase" title="Learn more about Reverse Transcriptase">reverse transcriptase</a>&nbsp;and integrate into the genome. However, as the&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/x-linked-severe-combined-immunodeficiency" title="Learn more about X Linked Severe Combined Immunodeficiency">X-SCID</a>&nbsp;trial and other, more recent gene therapy trials have demonstrated, integrating viruses can be dangerous due to the risk of insertional&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/carcinogenesis" title="Learn more about Carcinogenesis">oncogenesis</a><a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0090" name="bbib0090">[18]</a>. In the OTC trial, by&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/contrast" title="Learn more about Contrast">contrast</a>,&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/adenoviridae" title="Learn more about Adenoviridae">adenoviruses</a>&nbsp;were fatal due to their high&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immunogenicity" title="Learn more about Immunogenicity">immunogenicity</a>. Various safety mechanisms have been proposed for both of these issues and are reviewed elsewhere&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0095" name="bbib0095">[19]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0100" name="bbib0100">[20]</a>.</p> <p>Another way to reduce the risks of insertional oncogenesis and immunogenicity is to use AAVs in place of AdVs. AAVs have a comparatively low incidence of integration in the genome and persist as&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/episome" title="Learn more about Episome">episomes</a>&nbsp;in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/primate" title="Learn more about Primate">primates</a>&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0105" name="bbib0105">[21]</a>. In addition, they cannot replicate without the aid of a&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/helper-virus" title="Learn more about Helper Virus">helper virus</a>. Although most of the population carries&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/antibody" title="Learn more about Antibody">antibodies</a>&nbsp;for some strains of AAV (AAV-1 and AAV-2),&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/prevalence" title="Learn more about Prevalence">prevalence</a>&nbsp;of antibodies for other strains such as AAV-5, AAV-6, AAV-8 and AAV-9 is much lower and therefore these alternatives may be suitable for gene therapy&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0110" name="bbib0110">[22]</a>. Combining AAVs with CRISPR-Cas9 promises a&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/solution-and-solubility" title="Learn more about Solution and Solubility">solution</a>&nbsp;which allows diverse and lasting gene editing with minimal immunogenic effects.</p> <p>AAVs are the smallest of viral vectors, with a&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genome-size" title="Learn more about Genome Size">genome size</a>&nbsp;of about 4.7&nbsp;kB.Therefore it can be difficult to package Cas9, with a&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/length" title="Learn more about Length">length</a>&nbsp;of about 4.2 kB, let alone its gRNAs. However, Cas9 can be split and then functionally reconstituted by, for example, adding auto-processing&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/intein" title="Learn more about Intein">intein</a>&nbsp;domains&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0115" name="bbib0115">[23]</a>. In fact an&nbsp;<em>in vitro</em>&nbsp;approach has been developed to modulate the expression level of full and truncated Cas9 protein by combining different domains in this way&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0120" name="bbib0120">[24]</a>. Furthermore, a recent study using AAV-9 to deliver split Cas9 in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mouse-model" title="Learn more about Mouse Model">mouse models</a>&nbsp;did not incite any&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immune-response" title="Learn more about Immune Response">immune response</a>&nbsp;to the viral vector itself, although antibodies were made against the Cas9 protein&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0125" name="bbib0125">[25]</a>.</p> <p>Lastly, alternatives to AAVs, AdVs and retroviruses are emerging. For instance,&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sendai-virus" title="Learn more about Sendai Virus">Sendai virus</a>&nbsp;is an attractive option as, unlike the main three viral vectors, it forms no DNA intermediate during its viral cycle. This means it has the least risk of integration into the genome. It has also been successfully used to deliver&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/crispr" title="Learn more about CRISPR">CRISPR</a>&nbsp;<em>in vitro</em><a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0130" name="bbib0130">[26]</a>. Although viral vectors have been shown to deliver CRISPR both&nbsp;<em>in vitro</em>&nbsp;and&nbsp;<em>in vivo</em>, whether this strategy will specifically destroy&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/neoplasm" title="Learn more about Neoplasm">tumors</a>&nbsp;in an&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/animal" title="Learn more about Animal">animal</a>&nbsp;model remains to be seen.</p> <h2>Non-viral vectors</h2> <p>The issues of safety,&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immunogenicity" title="Learn more about Immunogenicity">immunogenicity</a>, and payload size in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/virus-vector" title="Learn more about Virus Vector">viral vectors</a>&nbsp;have prompted research into alternative methods of&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/dna" title="Learn more about DNA">DNA</a>&nbsp;delivery&nbsp;<em>in vivo</em>.&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/non-viral-vector" title="Learn more about Non-Viral Vector">Non-viral vectors</a>&nbsp;made using&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/lipid" title="Learn more about Lipid">lipid</a>&nbsp;bilayers or cationic&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/polymer" title="Learn more about Polymer">polymers</a>&nbsp;such as&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/polyethyleneimine" title="Learn more about Polyethyleneimine">polyethyleneimine</a>(PEI) make up a class of synthetic vectors which have long been used to deliver DNA to cells&nbsp;<em>in vitro</em>, and generally have larger genetic payloads and fewer&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/toxicity" title="Learn more about Toxicity">toxic effects</a>&nbsp;compared to viral vectors&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0135" name="bbib0135">[27]</a>. Unlike with viral vectors, synthetic vectors do not contain immunogenic pathogen-associated&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/molecule" title="Learn more about Molecule">molecules</a>, and patients are unlikely to have pre-existing&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/immunity" title="Learn more about Immunity">immunity</a>&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0140" name="bbib0140">[28]</a>. In spite of these advantages, synthetic vectors were used in less than 6% of gene therapy trials as of 2012&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0065" name="bbib0065">[13]</a>. The primary disadvantage of synthetic vectors is their low&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/transfer-gene" title="Learn more about Transfer Gene">gene transfer</a>efficiency compared to viral vectors, which limits their utility for both systemic and intratumoral delivery&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0145" name="bbib0145">[29]</a>. Advances in the design of lipid and&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/polymerization" title="Learn more about Polymerization">polymeric</a>vectors have increased the feasibility of delivering DNA and&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/rna" title="Learn more about RNA">RNA</a>&nbsp;for therapy&nbsp;<em>in vivo</em>&nbsp;(reviewed in Ref.&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0135" name="bbib0135">[27]</a>); however, delivery of CRISPR-Cas9 presents its own unique difficulties.</p> <p>Although synthetic lipid or polymer&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/nanoparticle" title="Learn more about Nanoparticle">nanoparticles</a>&nbsp;can easily be made to carry a payload much larger than 4.2&nbsp;kb, which is the size of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cas9" title="Learn more about Cas9">Cas9</a>, nanoparticles must have a small&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/volume" title="Learn more about Volume">volume</a>&nbsp;in order to be delivered systemically through the endothelial gaps in blood vessels&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0145" name="bbib0145">[29]</a>. This means that the concentration of DNA inside of the nanoparticles must be very high in order to deliver CRISPR-Cas9 intravenously&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0150" name="bbib0150">[30]</a>. In addition, as with viral vectors, delivery of DNA encoding CRISPR-Cas9 runs the risk of random integration into the genome&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0155" name="bbib0155">[31]</a>.&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/plasmid" title="Learn more about Plasmid">Plasmid</a>&nbsp;delivery can increase the risk of non-specific as the plasmids persist in the cell long enough for these off-target effects to take place&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0160" name="bbib0160">[32]</a>. To address these problems, several recent studies have utilized non-viral vectors to deliver Cas9&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/protein" title="Learn more about Protein">protein</a>&nbsp;rather than DNA&nbsp;<em>in vivo</em>&nbsp;which leads to more&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/transient-expression" title="Learn more about Transient Expression">transient expression</a>&nbsp;and does not cause insertional&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/carcinogenesis" title="Learn more about Carcinogenesis">oncogenesis</a>&nbsp;via genome integration. Delivery of Cas9 protein in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mouse" title="Learn more about Mouse">mouse</a>&nbsp;models using lipid and polymeric nanoparticles has proven especially promising.</p> <p>Cas9 has recently been successfully delivered to&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mammalian-cell" title="Learn more about Mammalian Cell">mammalian cells</a>&nbsp;using cationic lipid nanoparticles (liposomes)&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0160" name="bbib0160">[32]</a>. Although Cas9 itself is not sufficiently anionic for delivery with cationic lipids, complexing Cas9 with a gRNA molecule increased the negative charge on the protein&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0160" name="bbib0160">[32]</a>.&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/transfection" title="Learn more about Transfection">Transfection</a>&nbsp;of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/human" title="Learn more about Human">human</a>&nbsp;cells&nbsp;<em>in vitro</em>&nbsp;showed that&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/protein-delivery" title="Learn more about Protein Delivery">protein delivery</a>&nbsp;was slightly more efficient than plasmid delivery, and resulted in a tenfold decrease in nonspecific&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genome-editing" title="Learn more about Genome Editing">genome editing</a>. Injection of the Cas9:gRNA:lipid complex into the mouse inner ear resulted in 20% transfection of mouse inner ear cells, with no detectable toxicity&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0160" name="bbib0160">[32]</a>. A follow-up study was able to improve the transfection efficiency by utilizing bioreducible lipids, which degrade in the reductive environment of the cell, allowing the cargo to be released after endosomal&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/escape-behavior" title="Learn more about Escape Behavior">escape</a>&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0165" name="bbib0165">[33]</a>. Using this method, the transfection efficiency of human cells with CRISPR-Cas9&nbsp;<em>in vitro</em>was increased from approximately 40% to over 70%&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0160" name="bbib0160">[32]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0165" name="bbib0165">[33]</a>. Although&nbsp;<em>in vivo</em>editing with CRISPR-Cas9 was not attempted, the lipid nanoparticles were used to deliver a different genome editing&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/cre-recombinase" title="Learn more about Cre Recombinase">protein, Cre</a>&nbsp;recombinase to mouse brain cells near the site of injection&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0165" name="bbib0165">[33]</a>.</p> <p>Nanoparticles made of&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/palindromic-dna" title="Learn more about Palindromic DNA">palindromic DNA</a>, known as &lsquo;nanoclews&rsquo;, have also been used to deliver CRISPR-Cas9 to&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/neoplasm" title="Learn more about Neoplasm">tumor</a>&nbsp;cells&nbsp;<em>in vivo</em>. The nanoclews were complexed with the cationic polymer PEI to offset the negative charge of the DNA backbone&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0170" name="bbib0170">[34]</a>. In this study, intratumoral injection of nanoclews carrying Cas9 protein and gRNA in mice resulted in 25% transfection of tumor cells 10&nbsp;days after injection. This is comparable to the transfection efficiency observed by Zuris&nbsp;<em>et al</em>. in the mouse inner ear; however, it is unclear whether 20&ndash;25% transfection efficiency will be sufficient to cause&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/tumor-regression" title="Learn more about Tumor Regression">tumor regression</a>.</p> <p>For systemic delivery of CRISPR-Cas9, zwitterionic amino lipids (ZALs) were optimized to carry very long RNA molecules&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0150" name="bbib0150">[30]</a>. Separate ZAL nanoparticles carrying Cas9 mRNA and gRNA, respectively, were administered to mice intravenously, resulting in gene editing in liver, lung, and kidney tissues. Quantification of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gene-expression" title="Learn more about Gene Expression">gene expression</a>&nbsp;in the liver revealed that up to 3.5% of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/hepatocyte" title="Learn more about Hepatocyte">hepatocytes</a>&nbsp;had been successfully edited with CRISPR-Cas9&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0150" name="bbib0150">[30]</a>. These results indicate that lipid nanoparticles can be used to deliver CRISPR-Cas9 systemically; however, these nanoparticles were not targeted to any specific tissue, and therefore transfected several tissues with relatively low efficiency. To target&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/malignant-neoplasm" title="Learn more about Malignant Neoplasm">cancer</a>&nbsp;systemically, synthetic nanoparticles need to be designed to specifically transfect cancer cells. Nanoparticles made from a variety of different materials are available in order to achieve this&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/motivation" title="Learn more about Motivation">goal</a>&nbsp;(reviewed in Ref.&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0175" name="bbib0175">[35]</a>).</p> <p>For the treatment of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/solid" title="Learn more about Solid">solid</a>&nbsp;tumors, an alterative to systemic injection is local delivery using hydrogels: scaffolds formed out of naturally occurring or synthetic biocompatible polymers, which can be complexed with DNA, RNA or protein&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0180" name="bbib0180">[36]</a>. Hydrogels can be inserted during surgery or injected with a needle near the site of injection, providing local and&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/sustained-drug-release" title="Learn more about Sustained Drug Release">sustained release</a>&nbsp;of therapeutic&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/nested-gene" title="Learn more about Nested Gene">genes</a><a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0185" name="bbib0185">[37]</a>.The hydrogels themselves induce very little toxicity in healthy tissues, and are poorly immunogenic&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0180" name="bbib0180">[36]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0185" name="bbib0185">[37]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0190" name="bbib0190">[38]</a>. Although this strategy has not yet been applied to&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/crispr" title="Learn more about CRISPR">CRISPR</a>, several studies have utilized hydrogels to deliver RNAs for cancer treatment. Gold or synthetic nanoparticles coated with siRNA or miRNA and loaded onto an injectable hydrogel scaffold were shown to cause tumor&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/reduction-chemistry" title="Learn more about Reduction (Chemistry)">reduction</a>&nbsp;and prevent macro-metastases&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0185" name="bbib0185">[37]</a>,&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0195" name="bbib0195">[39]</a>. Finally,&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/gold-nanoparticle" title="Learn more about Gold Nanoparticle">gold nanoparticles</a>have been replaced with with synthetic particles made from poly(beta-amino ester), which dissolves in the acidic&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/tumor-microenvironment" title="Learn more about Tumor Microenvironment">tumor microenvironment</a>, facilitating release of the payload&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0190" name="bbib0190">[38]</a>. By coating these nanoparticles with siRNA and loading them into an injectable hydrogel, the authors were able to achieve 70%&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gene-silencing" title="Learn more about Gene Silencing">gene silencing</a>in&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/murine" title="Learn more about Murine">murine</a>&nbsp;tumors compared with 20% using intravenously injected nanoparticles&nbsp;<a href="https://www.sciencedirect.com/science/article/pii/S1740675717300026#bib0190" name="bbib0190">[38]</a>. These results suggest that hydrogels could be used to aid the delivery of CRISPR-Cas9 by synthetic nanoparticles.</p> <p>We have seen that a variety of synthetic vectors including&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/liposome" title="Learn more about Liposome">liposomes</a>, bioreducible lipids, nanoclews, ZALs and hydrogels show promise for delivery of CRISPR-Cas&nbsp;<em>in vitro</em>. Overall, studies utilizing synthetic vectors to treat cancer using CRISPR-Cas9&nbsp;<em>in vivo</em>&nbsp;must be carried out before the&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/electric-potential" title="Learn more about Electric Potential">potential</a>&nbsp;of non-viral vectors can be realized.</p> <h2>Conclusion</h2> <p><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/genome-editing" title="Learn more about Genome Editing">Genome editing</a>&nbsp;with CRISPR-Cas has revolutionized the field of gene therapy in just a few short years. However, the specific limitations of CRISPR-Cas9, including the large size of&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cas9" title="Learn more about Cas9">Cas9</a>&nbsp;and its&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/electric-potential" title="Learn more about Electric Potential">potential</a>&nbsp;for off-target effects, pose challenges to vector design. As with other forms of gene therapy the primary concerns include efficiency and specificity of delivery. Here we have described very recent&nbsp;<em>in vitro</em>&nbsp;and pre-clinical work done to deliver CRISPR-Cas9 using both viral and non-viral methods, as well as the benefits and downsides associated with each delivery method.&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/virus-vector" title="Learn more about Virus Vector">Viral vectors</a>&nbsp;are very efficient, but safety issues have limited their usage and their payload is small.&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/non-viral-vector" title="Learn more about Non-Viral Vector">Non-viral vectors</a>&nbsp;are being rapidly developed at present, especially&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/nanoparticle" title="Learn more about Nanoparticle">nanoparticles</a>. These nanoparticles can deliver either Cas9&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/protein" title="Learn more about Protein">protein</a>&nbsp;or mRNA, however, their&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/transfection" title="Learn more about Transfection">transfection</a>&nbsp;efficiency is still much lower compared to viral vectors. We have highlighted potential methods to surmount the&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/weakness" title="Learn more about Weakness">weaknesses</a>&nbsp;of each method. When brought into the clinic these&nbsp;<a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/development" title="Learn more about Development">developments</a>&nbsp;will allow CRISPR-Cas9 to target&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/malignant-neoplasm" title="Learn more about Malignant Neoplasm">cancer</a>&nbsp;efficiently throughout the body without harmful&nbsp;<a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/side-effect" title="Learn more about Side Effect">side effects</a>, allowing the enormous potential of cancer gene therapy to be realized.</p>

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