The first attempt in the United States to use a gene editing tool called CRISPR against cancer

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The first attempt in the United States to use a gene editing tool called CRISPR against cancer seems safe in the three patients who have had it so far, but it’s too soon to know if it will improve survival, doctors reported Wednesday.

The doctors were able to take immune system cells from the patients’ blood and alter them genetically to help them recognize and fight cancer, with minimal and manageable side effects.

The treatment deletes three genes that might have been hindering these cells’ ability to attack the disease, and adds a new, fourth feature to help them do the job.

“It’s the most complicated genetic, cellular engineering that’s been attempted so far,” said the study leader, Dr. Edward Stadtmauer of the University of Pennsylvania in Philadelphia. “This is proof that we can safely do gene editing of these cells.”

After two to three months, one patient’s cancer continued to worsen and another was stable. The third patient was treated too recently to know how she’ll fare.

The plan is to treat 15 more patients and assess safety and how well it works.

“It’s very early, but I’m incredibly encouraged by this,” said one independent expert, Dr. Aaron Gerds, a Cleveland Clinic cancer specialist.

Other cell therapies for some blood cancers “have been a huge hit, taking diseases that are uncurable and curing them,” and the gene editing may give a way to improve on those, he said.

Gene editing is a way to permanently change DNA to attack the root causes of a disease. CRISPR is a tool to cut DNA at a specific spot.

It’s long been used in the lab and is being tried for other diseases.

This study is not aimed at changing DNA within a person’s body.

Instead it seeks to remove, alter and give back to the patient cells that are super-powered to fight their cancer – a form of immunotherapy.

Chinese scientists reportedly have tried this for cancer patients, but this is the first such study outside that country.

It’s so novel that it took more than two years to get approval from U.S. government regulators to try it.

The early results were released by the American Society of Hematology; details will be given at its annual conference in December.

The study is sponsored by the University of Pennsylvania, the Parker Institute for Cancer Immunotherapy in San Francisco, and a biotech company, Tmunity Therapeutics. Several study leaders and the university have a financial stake in the company and may benefit from patents and licenses on the technology.

Two of the patients have multiple myeloma, a blood cancer, and the third has a sarcoma, cancer that forms in connective or soft tissue. All had failed multiple standard treatments and were out of good options.

Their blood was filtered to remove immune system soldiers called T cells, which were modified in the lab and then returned to the patients through an IV. It’s intended as a one-time treatment. The cells should multiply into an army within the body and act as a living drug.

So far, the cells have survived and have been multiplying as intended, Stadtmauer said.

“This is a brand new therapy” so not it’s not clear how soon any anti-cancer effects will be seen. Following these patients longer, and testing more of them, will tell, he said.


The CRISPR/Cas9 system

CRISPR/Cas9 is a prokaryotic, adaptive immune system that consists of a programmable RNA molecule that helps guide an associated Cas9 endonuclease to specific exogenous genetic invaders based on recognized sequences.1 

The CRISPR-Cas9 system consists of two components, a Cas9 endonuclease and a single-stranded guide RNA (sgRNA).2,3 The sgRNA directs the Cas9 endonuclease to cleave both DNA strands in a sequence-specific manner (Fig. ​(Fig.1).1).

DNA cleavage occurs at a sequence 3 base pairs upstream of an “NGG” protospacer adjacent motif (PAM).4

 Following the double-strand break (DSB), the genome is repaired by DNA-DSB repair mechanisms. Using the CRISPR/Cas9 system, targeted genome modifications can be made, such as the introduction of small insertions and deletions (indels) mediated through the relatively error-prone non-homologous end-joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR) pathway.5 Genes of interest can be easily targeted using a 17–21 nucleotide-targeting sequence.

To identify genes that are important for a particular phenotype, a pooled population of sgRNAs can be introduced into Cas9-expressing cells by phenotype-based screening of genomic changes.6 

In this review, we provide examples of current applications of this technology and speculate on future applications in cancer biology and oncology.

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Fig. 1
CRISPR/Cas9-based gene modification. Common methods of delivering the CRISPR system include a plasmid-based method and Cas9 protein complex with sgRNA or RNP. After the sgRNA binds to the target site of genomic DNA, the Cas9 protein creates a DSB around the PAM site. Random indels or precise modifications introduced into the genomic DNA by the NHEJ or HDR pathway

CRISPR/Cas9 variations

Many variations of the CRISPR/Cas9 system have been developed (Table ​(Table1).1). The Cas9 protein consists of a bi-lobed architecture and the sgRNA is captured between the alpha-helical and nuclease lobes.

In the nuclease lobe are two functional domains, HNH and RuvC. The RuvC domain belongs to the retroviral integrase superfamily of proteins and it cleaves the non-target DNA strand whereas the HNH domain cuts the targeted strand of the specific DNA. Normally, the HNH and RuvC domains generate a DSB.7 

The inactivation of both domains by a mutation at H840A and D10A in the HNH and RuvC domains, respectively, results in a catalytically inactive Cas9 (dCas9). However, a single mutation of HNH or RuvC results in the generation of a single-strand break rather than a DSB.

The Cas9 H840A and D10A mutants also have nickase activity wherein the RuV mutant D10A nicks the targeting strand and the HNH mutant H840A nicks the non-targeting strand. Because dCas9 is enzymatically inactive, it cannot cleave DNA.

However, it retains its RNA-guided DNA binding ability, which has led to several innovative applications.8 dCas9, when fused to a transcriptional repressor peptide such as KRAB (Kruppel associated box), can be used to knockdown gene expression by guiding RNA.

This fusion system can block the initiation of transcription and elongation and is referred as CRISPRi. The dCas9-KRAB fusion protein, when co-expressed with a target-specific sgRNA, binds the sgRNA, and the entire complex binds to the DNA strand, blocking the initiation of transcription and elongation resulting in depletion of transcripts of interest.9

 In a similar approach, dCas9 can also be used to activate gene expression if it is fused with an activator peptide such as the VP64 and VPR activation domains.

This complex is called CRISPRa and can increase transcription of target gene transcripts. CRISPRi and CRISPRa provide new tools for investigating human genome functions, transcriptome research, and regulation of functional factors in cancer biology and oncology.

This differs from the canonical CRISPR system that often causes meaningless mutations or leads to a chaotic phenotype.10 Compared with other CRISPR approaches, dCas9-based CRISPRi and CRISPRa are inducible, reversible, have fewer off-target effects, and low toxicity. These approaches have advantages in long non-coding (lnc) RNA knockdown and overexpression.11

 In cancer research, precise regulation of gene expression is a very useful approach and scientists have developed and expanded different systems, such as RNAi (RNA interference) and ORF (open reading frame) expression for loss or gain-of-function studies.11 RNAi has played a critical role in biological studies mainly because it has deterministic outcomes and is easy to deliver into mammalian cells.

On the other hand, CRISPR system-based tools are often difficult to deliver into mammalian cells. Two components are required compared with a single component RNAi. However, RNAi can also lead to unpredicted non-specific toxicity and strong siRNA/shRNA can cause extensive off-target effects.12

 Thus, even though CRISPR has disadvantages, CRISPR-based loss-of-function approaches are widely used because CRISPRi shows less endogenous off-target effects compared to RNAi, and also provides a high specificity of gene knockdown by blocking distinct promoters. It can also be useful for targeting lncRNA, whereas RNAi may be inefficient.13

Table 1

Variations of the CRISPR system

VariationsFeaturesEffectsAdvantagesDisadvantagesApplications in cancer research
CRISPR/Cas9WT Cas9; sgRNADouble-strand break at the target siteVersatile; effective; stable; easy accessibilityOff-target; PAM limited; different modified allelesSet up research model; functional gene study; drug target identification
CRISPR/Cas9 NickaseMutant Cas9 H840A or D10A; sgRNASingle-strand breakConvenient; efficient; flexible; precise, scalable; robustPAM limited;
2 sgRNA for KO
Manipulate epigenetic modifications; Simultaneous activation and repression
CRISPRidCas9; repressor peptide; sgRNABlock transcription elongation or knockdown transcriptsInducible; reversible;
low off-target effects;
low toxicity
PAM limited; off-target effects at bidirectional promotersGenome and transcriptome research;
LncRNA knockdown and overexpression
CRISPRadCas9; activator peptide; sgRNAIncrease transcriptionPAM limited;
complicated to deliver the multiple components

Applications of CRISPR/Cas9 in cancer research

Generation of cancer models

Cancers are driven by processes influenced by underlying genes.22 Being able to decipher the molecular genetics of disease is crucial to elucidate the underlying mechanisms.23 Cell lines and animal models are invaluable for dissecting the relationship of the genotype, chemotherapeutic effects, and immune microenvironment. CRISPR genetically engineered cancer models now can be produced rapidly, efficiently, and inexpensively24 (Table ​(Table2).2). Leukemia models were generated by targeting several inactivated genes through a lentivirus-delivered Cas9–sgRNA system in primary hematopoietic stem and progenitor cells (HSPCs).25 The pooled lentiviruses target several genes, including Tet2, Runx1, Dnmt3a, Nf1, Ezh2, and Smc3. With a fluorescent marker, multiple targeted HSPCs were selected that are involved in the development of myeloid malignancy. The CRISPR/Cas9 technology has been used to generate several other cancer models.26 By introducing mutations of APCSMAD4TP53KRAS, and/or PIK3CA, an organoid model of colon cancer was built with CRISPR technology.27 In the future, the generation of precision cancer models would greatly stimulate the study of functional cancer genomics and enhance the development of precision cancer medicine.28

Table 2

CRISPR applications in cancer research

ApplicationTargetssgRNA designVehicle for deliveryFeaturesAdvantagesDisadvantages
Generate cancer modelHSPCs; healthy human organoidsTargeting the model type-related suppressors oncogenesPooled lentivirusDisrupt suppressors or edit oncogenesRapid, efficient, and inexpensiveSpecial delivery techniques; tissue limited
Synergistic gene studyCellsTargeting optional drug target from databaseLenti-double sgRNA libraryTogether with deep sequencingEffective, low cost, innovative approachDouble sgRNA construction; need highly efficient sgRNA; special analysis
Target validationDrug or anticancer reagent resistant cellsLentiviral library from Addgene; or optional targetsPlasmidIdentify the target from resistant cells by sequencingEffectiveFalse-positives
Gene diagnoseGenomeTarget sensitive genesLentivirusTogether with Cas13a or Cas12a to induce collateral effectsSensitive, rapid, low costCertain template concentration

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Synergistic gene studies using CRISPR/Cas9

The CRISPR/Cas9 system also provides an effective strategy for the identification of synergistic gene interactions, which could be used to block drug resistance. A CRISPR-based double knockout (CDKO) system in K562 leukemia cells has been developed using a double sgRNA library system to screen for combinatorial genes and identify pairs of synthetic lethal drug targets. Together with deep sequencing, phenotype measurement and gene analysis have identified interactions between synergistic drug targets, like BCL2L1 and MCL1.29 Another simple and efficient strategy called CombiGEM (combinatorial genetics en masse)-CRISPR was also developed to analyze combinatorial gene function.30 It is similar to the CDKO system in which two pooled sgRNA libraries were combined in one vector. With this approach, some genetic hits (e.g., KDM6B + BRD4) were discovered. Depleting these genes using the CombiGEM system has shown stronger synergistic efficacy against ovarian cancer cell proliferation compared to a reported small-molecule inhibitor.30 Compared to drug inhibition, the CRISPR system costs less.9 The power of the CRISPR library to screen functional variants could thus play an important role in precision cancer medicine.31 Indeed, this innovative approach could allow for the development of personalized genotype-based therapies built upon genotype-specific targets.32

Functional gene screening using CRISPR/Cas9

Precision cancer medicine has resulted in the development of many targeted drugs to treat different cancers. Targeted therapy already has shown enormous potential but several challenges still exist. Only patients who exhibit a certain mutation or altered gene expression respond to the targeted drug treatment and drug resistance to the therapy still occurs.33 

Functional genome-screening approaches using the CRISPR system could reveal gene expression changes after treatment and pinpoint genes associated with resistance to the targeted drugs, thereby identifying new biomarkers for precision therapy and providing new insights into cancer development.34 

One successful example involved screening for a cancer metastasis-related gene with a CRISPR-Cas9-mediated loss-of-function screen.35 This group infected a non-metastatic lung cancer cell line with a mouse genome-scale CRISPR knockout (mGeCKO) sgRNA library and the transduced cells were subcutaneously transplanted into immunocompromised mice. Six weeks later, the mice exhibiting lung cancer metastasis were selected for sequencing the enriched sgRNA.

Finally, several candidate genes associated with lung metastasis were identified and validated, including the already reported genes Pten,36 miR-152,37 and miR-345,38 and several new genes like Nf2, Trim72, and Fga.

Other loss-of-function screens were also applied to examine suppressor genes in liver tumors.39 In this study, p53−/−/Myc mouse embryonic liver progenitor cells were infected with an mGeCKO library and transplanted into nude mice. sgRNAs that were increased 8-fold were chosen as candidates and Nf1Plxnb1Flrt2, and B9d1 were identified as new tumor suppressors involved in liver cancer formation. Another group applied CRISPR interference (CRISPRi) to screen for functional lncRNA loci, which could modify cell growth.40 They designed a comprehensive sgRNA library to target the lncRNA transcription start site (TSS). The library was transduced into several different cell lines and together with sequence analysis, 499 lncRNAs were identified as associated with cell growth.

CRISPRi can repress the transcription of targeted genes by recruiting the complex of dCas9 and a repressor to the TSS, which is more suitable for lncRNA gene research.10 In summary, combining CRISPR-based functional genetic screening is a powerful approach to validate alternative genes associated with a specific phenotype.41

Target validation by CRISPR/Cas9

Revealing the mechanism of action for small-molecule drugs is a time-consuming and laborious process.42 Efficiently identifying new drug targets with a CRISPR-Cas-based genetic screening system (CRISPRres) containing large sgRNA libraries is now possible.43 If the molecular binding site is depleted or mutated, cancer cells commonly acquire resistance, but the molecular target could be clearly identified by sgRNA sequencing.

CRISPR was used to successfully identify nicotinamide phosphoribosyl transferase as the primary target of KPT-9274, an anticancer agent. Based on CRISPR/Cas9 technology, another team set up a system called DrugTargetseqR to identify direct physiologic targets by mutating potential targets for drug resistance.44 In this study, kinesin-5 was confirmed as the direct target of ispinesib by mutating kinesin-5 D130V or A133P in HeLa cells.

Targeting the exons that encode functional protein domains rather than the 5′ exons reportedly is a better method using the CRISPR system for identifying drug targets.45 In this case, a negative selection system was constructed with a GFP reporter and used to screen hundreds of chromatin regulatory domains in leukemia cells.

The sgRNAs targeting of functional domains, like the ATPase domain or DNA-binding domain, led to a stronger negative selection phenotype compared to targeting the 5′ exon. Obviously, combining CRISPR genome-editing with deep sequencing or cellular biophysical assays could produce new insights into target validation studies.46,47

Gene diagnosis

Genetic diagnostics to determine sensitive genes is critical for cancer prevention.48 Although a low frequency mutation is not easily determined by sequencing, a CRISPR-based diagnostic system called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), has been established.49 

A key factor in this system is Cas13a, an RNA-guided RNase, which induces robust non-specific single-stranded DNA (ssDNA) trans-cleavage as a collateral effect.50 

Another essential element is the reporter signal, which is released after RNA cleavage. This method has been used to detect two cancer mutants, BRAF V600E and EGFR L858R, and appears to be a highly sensitive detection approach.

A similar system referred to DETECTR (DNA endonuclease-targeted CRISPR trans reporter) has been developed.51 In this system, another Cas family member, Cas12a, is used, and acts similar to Cas13a. An additional enzyme, RPA (recombinase polymerase amplification), is used to amplify micro-samples. RPA can be used as a detection tool for screening for infections in cancers. The system was used to detect HPV types 16 and 18 in lung carcinomas and appears to be a rapid and inexpensive approach.52

Anticancer applications in clinical trials

Based on promising results of pre-clinical studies, the CRISPR/Cas9 system could also potentially be used clinically to target cancer-causing genes. At this time, eleven clinical trials are underway testing the effectiveness of CRISPR for cancer therapies (Table ​(Table3).3). Seven of the eleven trials are immunotherapies that target program cell death-1 (PD-1) protein expression. The PD-1 protein and programmed cell death ligands (PD-Ls) are important for the negative regulation of the immune system, specifically on T-cells. Their attenuation of the immune response helps tumor cells survive by evading the immune system.53 

Pembrolizumab, a monoclonal antibody against PD-1, confirmed that blocking PD-1 and PD-L1 in the immune system could significantly increase the overall survival rate in cancer patients.54 PD-1 is thus an attractive target for immunotherapy and PD-1 inhibitors have been recently approved by the U.S. Food and Drug Administration (FDA) for cancer immunotherapy.

Coincidentally, a team in China has gone one step further by using CRISPR/Cas9 to directly target PD-1 in patients (NCT02793856). Using CRISPR/Cas9, they disabled PD-1 expression in cells harvested from a metastatic non-small-cell lung cancer patient.

They expanded the cells in a large culture system and then injected the modified cells back into the patient.55 Based on the results of a dose-escalation study, the safety of PD-1 knockout-engineered T-cells in treating metastatic non-small cell lung cancer will be evaluated.

Similar trials targeting PD-1 expression in T-cells are being conducted in prostate (NCT02867345), bladder (NCT02863913), and renal cell cancers (NCT02867332). Another phase II clinical study has applied the same PD-1 knockout on T-cells for esophageal cancer (NCT03081715).

Table 3

Anticancer applications in clinical trials

ApplicationsTarget siteStudy phaseEditing strategyClinical trials identification
Advanced esophageal cancerPD-1Phase IIPD-1 knockoutNCT03081715
Castration resistant prostate cancerPD-1Phase IPD-1 knockoutNCT02867345
Muscle-invasive bladder cancerPD-1Phase IPD-1 knockoutNCT02863913
Metastatic non-small cell lung cancerPD-1Phase IPD-1 knockoutNCT02793856
EBV associated malignanciesPD-1Phase I
Phase II
PD-1 knockoutNCT03044743
Metastatic renal cell carcinomaPD-1Phase IPD-1 knockoutNCT02867332
Relapsed or refractory leukemia and lymphomaCD19 and CD20 or CD22Phase I
Phase II
Edit CD19 and CD20 or CD22NCT03398967
Human papillomavirus-related malignant neoplasmHPV16-E6/E7
HPV18 E6/E7
Phase IHPV16-E6/E7 or HPV18 E6/E7
knockout
NCT03057912
CD19 + leukemia and lymphomaTCR
B2M
Phase I
Phase II
TCR and B2M
knockout
NCT03166878
Tumor of the central nervous systemNF1Fix NF1 mutation alleleNCT03332030
Multiple myeloma
Melanoma
Synovial sarcoma
Myxoid/round cell liposarcoma
TCR
PD-1
Phase ITCR and PD-1 knockoutNCT03399448

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Generating chimeric antigen receptor (CAR) T-cells by CRISPR/Cas9 is another ex vivo approach in clinical trials. Researchers from the University of Pennsylvania organized the first-in-human trial to test the effect of HLA-A*0201 restricted NY-ESO-1 redirected engineered T-cells in a wide range of cancer types, including relapsed refractory multiple myeloma (MM), melanoma, synovial sarcoma, and myxoid/round cell liposarcoma (NCT03399448). Tumor rejection activity might be enhanced by eliminating endogenous TCR and PD-1 with the use of CRISPR.

Another clinical trial (NCT03166878) focused on CD19+ leukemia and lymphoma. Allogeneic CD19-directed CAR T-cells were generated using the lentiviral delivery of the CAR receptor and CRISPR RNA by electroporation to disrupt the endogenous TCR and B2M genes.

This approach might assist in evading host-mediated immunity and thus deliver anti-leukemic effects to patients without having to worry about graft-versus-host-disease (GVHD).

Unfortunately, a subset of patients relapse due to the loss of CD19 in tumor cells. Thus another clinical trial is focusing on CRISPR-edited dual specificity CD19 and CD20 or CD22 CAR T-cells, which could recognize and kill the CD19-negative malignant cells through recognition of CD20 or CD22 (NCT03398967).

This may be a complementary approach for a wide range of patients. In another application, CRISPR/Cas9 was used to disrupt the human papillomavirus-16 (HPV16) E7 protein, an oncogenic protein that is important for the maintenance of the malignant phenotype in cervical cancer.

When E7 was disrupted in HPV-positive cervical cancer cells, inhibition of tumor growth inhibition, and induction of apoptosis occurred.56 These promising results have led to a phase I clinical trial (NCT03057912) to evaluate the safety and efficacy of TALEN-HPV E6/E7 and CRISPR/Cas9-HPV E6/E7 in treating HPV persistent and HPV-related cervical intraepithelial neoplasia. In this study, a CRISPR/Cas9 plasmid targeting HPV16-E6/E7T1 or HPV18 E6/E7T2 was administered twice a week for four weeks to disrupt the expression.

Another clinical trial using CRISPR technology has been designed to screen and identify drugs (NCT03332030). In this trial, an induced pluripotent stem cell (iPSC) bank was established from phenotypically well-characterized patients with Neurofibromatosis type 1 (NF1). NF1 is a frequent neurocutaneous syndrome which easily causes various benign or malignant tumors.57 To identify a NF1 specific target drug, CRISPR/Cas9 was used to develop different cell lines, NF1 wild type (NF1+/+), NF1 heterozygous (NF1+/−), and NF1 homozygous (NF1−/−). By examining the reverse or alleviated phenotypes, NF1-specific drugs might be identified. Although results from CRISPR/Cas9 clinical studies might be promising, more work is needed to assure that CRISPR/Cas9 is a safe and effective tool for treating human cancers.

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