Double Downside of HIV CRISPR therapy
Larry H. Bernstein, MD, FCAP, Curator
Hitting HIV with CRISPR/Cas9 Can Arouse Resistance
This visual abstract depicts how HIV-1 can escape Cas9/sgRNA-mediated inhibition. The researchers reveal that the NHEJ repair machinery generates mutations in the HIV-1 Cas9 cleavage site that result in two outcomes: viral replication suppression and viral escape. [Wang et al./Cell Reports] http://www.genengnews.com/Media/images/GENHighlight/thumb_Apr8_2016_CellReports_HIVCas90718814213.jpg
Stretches of DNA altered by the human immunodeficiency virus (HIV) can be targeted by the CRISPR/Cas9 endonuclease system, resulting in strategically placed cuts, imperfect repairs to those cuts, and—finally—the end of viral replication. But in some cases, the battle-scarred DNA that CRISPR/Cas9 leaves behind won’t give up the fight. Worse, this DNA becomes harder to recognize, by dint of its scars, and becomes even more dangerous. It acquires a form of resistance, the ability to duck renewed attacks from CRISPR/Cas9.
This finding emerged from a study carried out by an international team of scientists that represented McGill University, the University of Montreal, the Chinese Academy of Medical Sciences, and Peking Union Medical College. These scientists, led by McGill’s Chen Liang, Ph.D., found that when CRISPR/Cas9 is used to mutate HIV-1 within cellular DNA, two outcomes are possible: (1) inactivation of HIV-1 and (2) acceleration of viral escape. This finding, the researchers cautioned, potentially limits the use of CRISPR/Cas9 in HIV-1 therapy.
The researchers also sounded an optimistic note. They pointed to strategies that could help overcome HIV’s tendency to escape CRISPR/Cas9’s antiviral action. For example, targeting multiple sites with CRISPR/Cas9 or using other enzymes aside from Cas9. Once a solution is identified, the next barrier will be identifying ways to deliver the treatment to patients.
The research team’s work appeared April 7 in the journal Cell Reports, in an article entitled, “CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape.” The article emphasized the importance of the CRISPR/Cas9 system’s reliance on single guide RNA (sgRNA), the programmable element of the system that allows DNA to be cleaved at specific sequences.
“Using HIV-1, we have now demonstrated that many of [CRISPR/Cas9-derived mutations or indels] are indeed lethal for the virus, but that others lead to the emergence of replication competent viruses that are resistant to Cas9/sgRNA,” wrote the article’s authors. “This unexpected contribution of Cas9 to the development of viral resistance is facilitated by some indels that are not deleterious for viral replication, but that are refractory to recognition by the same sgRNA as a result of changing the target DNA sequences.”
The authors added that indels that are compatible with viral viability should be taken into consideration if Cas9/sgRNA is used to treat virus infection and genetic diseases. They expect that such indels would contribute to virus escape not only when Cas9/sgRNA is utilized to control new infections, but also in the context of eliminating latent viral DNA of herpes viruses, hepatitis B virus (HBV), and HIV, among others.
“CRISPR/Cas9 gives a new hope toward finding a cure, not just for HIV-1, but for many other viruses,” said Dr. Liang. “We have a long road toward the goal, and there may be many barriers and limitations that we need to overcome, but we’re confident that we will find success.”
CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape
- •Cas9/sgRNA suppresses HIV-1 replication
- •HIV-1 escapes from inhibition mediated by Cas9/sgRNA
- •Escape mutations are located to the Cas9 cleavage site within the target viral DNA
- •Cas9/sgRNA-induced mutations assist viral escape
Cas9 cleaves specific DNA sequences with the assistance of a programmable single guide RNA (sgRNA). Repairing this broken DNA by the cell’s error-prone non-homologous end joining (NHEJ) machinery leads to insertions and deletions (indels) that often impair DNA function. Using HIV-1, we have now demonstrated that many of these indels are indeed lethal for the virus, but that others lead to the emergence of replication competent viruses that are resistant to Cas9/sgRNA. This unexpected contribution of Cas9 to the development of viral resistance is facilitated by some indels that are not deleterious for viral replication, but that are refractory to recognition by the same sgRNA as a result of changing the target DNA sequences. This observation illustrates two opposite outcomes of Cas9/sgRNA action, i.e., inactivation of HIV-1 and acceleration of viral escape, thereby potentially limiting the use of Cas9/sgRNA in HIV-1 therapy.
HIV-1 Escapes from Suppression Mediated by Cas9/sgRNA
To investigate whether HIV-1 is able to escape from Cas9/sgRNA-mediated inhibition, we first generated CD4+ SupT1 cell lines that stably expressed both Cas9 and sgRNA that we previously showed could inhibit HIV-1 production in transient transfection experiments (Zhu et al., 2015). These Cas9 and sgRNA genes were stably transduced into SupT1 cells using a lentiviral vector (Sanjana et al., 2014). These Cas9/sgRNA-expressing cells showed growth capacity similar to that of the control cells (Figure S1A). The T4 sgRNA targets the overlapping open reading frames (ORFs) of HIV-1 gag/pol genes, while T10 targets the overlapping ORFs of HIV-1 env/rev genes (Figure 1A). Both viral targets are very conserved in HIV-1 sequences that are registered in the HIV database (Figure S1B). Since each of these two sgRNAs targets two specific viral genes, we conjectured that the genetic barrier should be high for HIV-1 to mutate and escape from inhibition. A control SupT1 cell line expressed Cas9 only.
We first tested these SupT1 cell lines by exposing them to the NL4-3 HIV-1 strain for a short term of infection. The results showed that T4 or T10 sgRNA together with Cas9 reduced the number of HIV-1 infected cells (Figure 1B) and diminished the production of infectious viruses (Figure 1C). To demonstrate that these reductions had resulted from the action of Cas9/sgRNA that causes indels, we extracted total cellular DNA from the infected cells, amplified the viral DNA region that was targeted by the T4 or T10 sgRNA, cloned the PCR products, and sequenced the DNA clones. Although no mutations were detected in the targeted viral DNA that was extracted from the infected control SupT1 cells, rich arrays of indels were identified in viral DNA from the infected SupT1 cells that expressed T4 or T10 sgRNA (Figures 1D and 1E). The percentages of indels for the T4 and T10 sgRNAs were approximately 25% and 30%, respectively. We also tested a number of these indels by inserting them into the HIV-1 DNA and observed that the majority of them abolished the production of infectious viruses in addition to the two substitution mutations that produced as much infectious viruses as the wild-type viral DNA did (Figure S1C). In addition to the NL4-3 HIV-1 strain, we further tested the T4 sgRNA against two primary HIV-1 isolates 89.6 and YU-2, as well as three transmitted founder viruses CH040, CH077, and CH106. The results showed that Cas9/T4 sgRNA caused indels in these viral DNA and strongly inhibited the production of each of these latter viruses (Figures S1D and S1E). Together, these results confirm that Cas9/sgRNA inhibits HIV-1 infection by introducing various mutations into viral DNA.
We next performed HIV-1 evolution experiments and monitored viral growth over prolonged times by measuring viral reverse transcriptase (RT) activity in culture supernatants. The results showed that HIV-1 replication was delayed in SupT1 cells expressing T4 or T10 sgRNA compared to viral replication in control SupT1 cells (Figure 2A). Nonetheless, viral production eventually peaked in the T4 and T10 SupT1 cells, showing that HIV-1 had escaped from suppression by Cas9/sgRNA. To further demonstrate viral escape, we collected viruses at the peaks of viral RT levels in the control, T4, and T10 cells, and then utilized the same RT levels of each virus to infect the corresponding SupT1 cell line. The T4 and T10 viruses displayed even moderately faster replication kinetics than the control virus in this second round of replication (Figure 2B), which suggests that the escape viruses might have gained mutations that improve viral infectivity.
The Cas9/sgRNA-Resistant HIV-1 Bears Mutations in the Viral DNA Region that Is Targeted by sgRNA
How HIV Can Escape an Experimental CRISPR Therapy
Targeting HIV-1 with CRISPR/Cas9 stops the virus from replicating, but can also help it escape, two recent studies show.
CRISPR/Cas9 gene editing has shown remarkable therapeutic potential, including the ability to fightpathogens like HIV. But the same process that inactivates the deadly virus may also enable it to escape the treatment, according to research led byChen Liang of McGill University in Montreal, published today (April 7) in Cell Reports.
“It’s very nice work which offers important information related to development and use of CRISPR/Cas9 for suppressing viruses—in this case, HIV infection,” neuroscientist Kamel Khalili of Temple University’s Lewis Katz School of Medicine in Philadelphia who was not part of the study told The Scientist. “Their data suggest targeting a single site within a viral gene can accelerate viral escape and emergence of mutant virus that remains resistant to initial targeting molecules.”
The findings essentially replicate those of another group, led by Atze Das of the Center for Infection and Immunity Amsterdam. The Das team’s findings appeared last month (February 16) in Molecular Therapy.
“We both demonstrated HIV-1 can be inhibited by the CRISPR/Cas system, and [that] the virus can escape,” Das, who was not involved in the new research, told The Scientist. He said the similarity of the studies was a coincidence.
A number of previous studies have demonstrated that CRISPR/Cas9 can be used to prevent HIV from replicating, but there wasn’t much evidence that the virus could escape that repression.
For the present study, Liang and colleagues used single guide RNAs (sgRNAs) and the Cas9 enzyme to target and snip out HIV-1 DNA from the genome of human T cells in vitro.
When Cas9 cuts the DNA, the cell repairs it using a process called nonhomologous end joining. This process is prone to errors, resulting in insertion and deletion mutations, or indels. By culturing cells with CRISPR-modified HIV, the researchers showed that these indels are lethal for the virus—they reduce the number of infected cells, and produce fewer infectious viruses.
However, some of the mutations were minor enough that the virus was able to escape and infect other cells. When the researchers cloned and sequenced the DNA from the escaped virus, they expected to see mutations throughout the DNA. “But we found that the mutations were all clustered at one site—where the Cas9 enzyme cleaves the viral DNA,” Liang told The Scientist. As a result, the sgRNA could no longer recognize the viral sequence, rendering it immune to future CRISPR attack.
The study provides “experimental evidence to show the existence of HIV viral escape for single guide RNA/Cas9,” neurovirologist Wenhui Hu of Temple University who was not involved in the work told The Scientist in an email, “although it was predicted and the proof of concept had been proposed or tested,” he added.
Liang’s team is now working on ways to address the problem. One method the authors suggest—demonstrated by Hu’s team and other groups—is to target the viral DNA using multiple guide RNAs, which increases the chances of disabling the virus.
We have shown that the indels generated by Cas9/sgRNA confer resistance against Cas9/sgRNA. Following recognition of PAM by Cas9, the adjacent target DNA unwinds and initially binds to the first 10-nt seed sequence of sgRNA (Jiang et al., 2015). Cas9 then cleaves the target DNA at a position three nucleotides away from PAM. The NHEJ machinery is then recruited to the double-stranded DNA break. While repairing this DNA lesion, NHEJ often introduces insertion or deletion mutations (Hsu et al., 2014). These indels result in a change in the target DNA sequence, thus preventing sgRNA from binding and leading to resistance to Cas9/sgRNA. If the sgRNA targets a viral DNA sequence that is not essential for viral replication, then the indels that are generated should quickly lead to the emergence of Cas9/sgRNA-resistant, replication-competent viruses, as we observed with the LTR-B sgRNA (Figure 4D). When essential viral genes are targeted by sgRNA, the resistance-conferring indels should contribute to viral escape if they minimally affect the functions of the targeted viral genes. These latter indels should maintain the ORFs of viral genes and lead to only minimal changes in numbers of amino acids (one or two). The results of our MiSeq experiments reveal that these types of indels do exist in transiently infected cells as well as in the escape viruses (Figures 4B and 4C). Results of our study do not exclude the possibility that, when cells contain two or more copies of proviral DNA, homologous repair may contribute to the generation of escape mutations. Our findings are corroborated by a recent report showing HIV-1 escapes from Cas9/sgRNA inhibition by mutating the sgRNA target sequence (Wang et al., 2016).
The indels that are compatible with viral viability should be taken into consideration if Cas9/sgRNA is used to treat virus infection and genetic diseases. We expect that such indels would contribute to virus escape not only when Cas9/sgRNA is utilized to control new infections, but also in the context of eliminating latent viral DNA of herpes viruses, HBV, and HIV, among others. This is because introduction of a viable indel into latent viral DNA should lead to the mutated viral DNA being resistant to Cas9/sgRNA, but still able to produce infectious viruses upon activation. One potential solution might be to simultaneously target two or multiple sites in the viral genome with an array of sgRNAs in the way that multiple siRNAs have been used to durably suppress HIV-1 replication (Schopman et al., 2010).
CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape
Molecular Therapy (2016); 24 3, 522–526. http://dx.doi.org:/10.1038/mt.2016.24
Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.
The clustered regularly interspaced short palindromic repeats-Cas9 system represents a versatile tool for genome engineering by enabling the induction of double-stranded breaks at specific sites in DNA.1 Sequence specificity is due to the gRNA that directs Cas9 to the complementary sequence present immediately upstream of a 3-nt protospacer adjacent motif in the target DNA. In mammalian cells, the double-stranded breaks can be repaired by the nonhomologous end-joining (NHEJ) pathway, which results in the frequent introduction of insertions, deletions, and nucleotide substitutions at the cleavage site, or by homology-directed repair, which depends on the presence of homologous DNA sequences.1,2
Several studies demonstrated that the Cas9/gRNA system can be used for inhibition of human pathogenic DNA viruses, including hepatitis B virus,3,4,5,6,7,8 Epstein–Barr virus,9 and human papilloma virus.10 Replication of retroviruses, like HIV-1, can also be inhibited with the Cas9/gRNA system by targeting the reverse-transcribed HIV-1 DNA replication intermediate or the proviral DNA upon integration into the cellular genome.2,11,12,13 Gene therapy approaches for the treatment of HIV-1 infected individuals have been proposed in which the Cas9 and antiviral gRNAs are directed to HIV-1 infected cells to inactivate or delete the integrated provirus, or in which blood stem cells are harnessed against new infections. However, Cas9/gRNA-mediated inhibition of virus production and/or replication has been shown only in short-term experiments, while we know that HIV-1 can escape from most if not all types of inhibitors, including small molecule antiviral drugs and sequence-specific attack by RNA interference. We therefore set out to identify viral escape strategies from Cas9/gRNA-mediated inhibition.
Design of gRNAs that effectively target the HIV-1 DNA genome
In silico algorithms were used to select 19 gRNAs that should target HIV-1 DNA with high efficiency and exhibit no off-target effects on cellular DNA (see Supplementary Table S1). Seven gRNAs were selected that target the long terminal repeat (LTR) region present at the 5′ and 3′ ends of the proviral genome (Figure 1a). Five of these (gLTR1–5) also target the accessory nef gene that overlaps the 3′ LTR, but that is not essential for in vitrovirus replication. Twelve gRNAs target sequences that encode other viral proteins, including well-conserved domains in the essential gag, pol and env genes and sequences of overlapping reading frames, like the tat and rev genes (Figure 1a). Nine selected gRNAs target sequences that are highly conserved among different HIV-1 isolates (Shannon entropy <0.2; gLTR7, gGag1, gGagPol, gPol1–4, gTatRev, and gEnv2), while the other gRNAs target less conserved HIV-1 domains (Shannon entropy ≥0.20; gLTR1–6, gGag2, gVpr, gEnv1, and gNef).
Cas9/gRNA targeting of the HIV-1 genome. (a) The HIV-1 proviral DNA with the position of gRNAs tested in this study. (b) The efficiency of gRNAs to silence HIV-1 DNA was tested in 293T cells transfected with plasmids expressing Cas9, gRNA, and HIV-1 LAI. To quantify viral gene expression, the viral capsid protein (CA-p24) was measured in the culture supernatant at 2 days after transfection. Average values (±SD) of four experiments are shown. Statistical analysis (independent samples’ t-test analysis) demonstrated that CA-p24 expression in the presence of antiviral gRNAs differed significantly from values measured with control gRNAs against luciferase and GFP (*P < 0.05).
We first tested the antiviral activity in transient transfections of 293T cells with plasmids expressing HIV-1, Cas9 and one of the anti-HIV gRNAs or control gRNAs targeting non-HIV sequences (luciferase, GFP). To quantify HIV-1 gene expression, we measured viral capsid protein (CA-p24) produced at 2 days after transfection (Figure 1b). A similar high CA-p24 level was observed when different control gRNAs were tested, but this level was significantly reduced for all anti-HIV gRNAs, which is likely due to Cas9/gRNA induced cleavage of the HIV-1 plasmid. Accordingly, the inhibitory effect was not observed in control experiments with only Cas9 or gRNA (data not shown). There may be some small differences in antiviral activity among the gRNAs, but we decided to move all inhibitors forward to antiviral tests in stably transduced T cells.
Inhibition of HIV-1 replication by the Cas9/gRNA system
SupT1 T cells were first transduced with a Cas9-expressing lentiviral vector. Stably transduced cells were selected and subsequently transduced with a lentiviral vector expressing one of the antiviral gRNAs. Of note, none of the selected gRNAs target the lentiviral vectors. Upon infection of transduced cells with the HIV-1 LAI isolate, virus replication was monitored by measuring the CA-p24 level in the culture supernatant. Efficient virus replication was apparent in control nontransduced SupT1 cells and in Cas9-only transduced cells, as reflected by a rapid increase in the CA-p24 level (Figure 2a) and the appearance of large virus-induced syncytia and cell death around day 10 after infection (Figure 2b; average time of HIV-1 breakthrough replication of four experiments are shown). HIV-1 replication in cells transduced with Cas9 and gRNAs targeting poorly conserved LTR sequences (gLTR1–6) was only marginally delayed (Figure 2a and data not shown) and breakthrough replication resulting in large syncytia was observed at 12–14 days (Figure 2b). Replication in cells transduced with Cas9 and gLTR7, which targets the highly conserved and essential TATA-box region of the LTR promoter, was more delayed and resulted in breakthrough replication at 19 days. A similar split was observed when targeting protein-coding regions. Targeting highly conserved HIV-1 sequences (gGag1, gGagPol, gPol1–4, gTatRev, and gEnv2) exhibits a more sustained antiviral effect (breakthrough replication in 20–43 days; Figure 2b) than targeting less conserved domains (gGag2, gVpr, gEnv1, and gNef; breakthrough replication in 11–17 days; Figure 2b). Surprisingly, despite their potency to suppress virus production (Figure 1b), some of the gRNAs inhibited virus replication only briefly and none prevented breakthrough virus replication. Moreover, the time required for breakthrough replication did not correlate with the potency of inhibiting HIV-1 production in 293T cells (see Supplementary Figure S1).
HIV-1 replication in Cas9 and gRNA expressing cells. (a,b) SupT1 cells stably transduced with Cas9 and gRNA expressing lentiviral vectors were infected with HIV-1 LAI. Virus replication was monitored by measuring the CA-p24 level in the culture supernatant (a) and by scoring the formation of virus-induced syncytia (b). The day at which massive syncytia were observed, which reflects breakthrough virus replication, is indicated. Average values of four experiments (±SD) are shown. SupT1, control nontransduced cells. SupT1-Cas9, cells transduced only with the Cas9 expressing vector. (c) Correlation between the level of inhibition (day of breakthrough replication; as shown in b and the conservation of target sequence amongst different HIV-1 isolates (Shannon entropy as shown in Supplementary Table S1). The Pearson’s correlation coefficient was calculated: r = −0.58.
The breakthrough viruses could represent viral escape variants that are no longer suppressed by the Cas9/gRNA system. Interestingly, the time required for breakthrough virus replication was longer for target sequences that are more conserved (Figure 2c: inverse correlation between the day of breakthrough replication and the Shannon entropy). Along these lines, the early escape observed for the gRNAs targeting nonconserved domains could be explained by many escape options that are available to the virus, whereas the relatively late escape observed for gRNAs targeting conserved domains could be due to the fewer escape options because important sequences are targeted. Nevertheless, the poor inhibition and very swift viral escape observed for some of the gRNAs is remarkable, as the evolutionary process underlying viral escape, i.e., the generation of sequence variation and subsequent outgrowth of variants with improved fitness, usually takes several weeks or even months, e.g., for RNA interference inhibitors tested in the same experimental system.14
NHEJ-induced mutations around the Cas9 cleavage site cause rapid HIV-1 escape
We first tested whether the breakthrough viruses were indeed resistant to the specific Cas9/gRNA set by passage onto fresh matching Cas9/gRNA SupT1 cells and control nontransduced cells. The breakthrough viruses replicated with similar efficiency on both cell lines (see Supplementary Figure S2), which confirmed the escape phenotype. Both cell lines were also infected with wild-type HIV-1 LAI, showing the selective replication block in restricted Cas9/gRNA cells.
CRISPR debate fueled by publication of second human embryo–editing paper
Xiangjin Kang, Wenyin He, Yuling Huang, Qian Yu, Yaoyong Chen,Xingcheng Gao, Xiaofang Sun, Yong Fan
Journal of Assisted Reproduction and Genetics 6 April 2016, pp 1-8
As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species.
The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos. Methods 3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos. Results By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations. Conclusions This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.