Adeno-associated Virus Integration Research Articles

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Generation of hepatitis C virus–resistant liver cells by genome editing–mediated stable expression of RNA aptamer

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. About 30% of these patients progress to cirrhosis within five years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR/Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a “safe harbor”, in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase (DFHR) minigene was precisely knocked-in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer-mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be utilized as a new alternative in the development of a treatment for HCV-induced liver diseases.

Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type–targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell–derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.

Genome Integration of Adeno-associated Virus (AAV) Vectors into Infected Cells

Genome Integration of Adeno-associated Virus (AAV) Vectors into Infected Cells

Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2-4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

BackgroundRedirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging.MethodsIn the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells.ResultsWe detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells.ConclusionLooking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis

BackgroundEngineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3’ end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results.ResultsWe collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients.By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported.We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients.ConclusionsOur findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

Abstract 2658: Oncogenicity of adeno-associated virus (AAV) integration into the genomes of liver cancer patients depends on the overlap between the AAV integration site and the AAV X gene

Abstract Viruses may affect human health by altering host immunity and the genome, contributing to chronic disease and cancer. The purpose of this study was to examine evidence of oncogenicity accompanying integrations of non-hepatitis viruses into the liver cancer genome through analysis of a cohort of 147 Thai liver cancer patients comprised of 47 hepatocellular carcinoma (HCC) cases and 100 intrahepatic cholangiocarcinoma (iCCA) cases. For each case, we generated paired RNASeq data from cancerous tissue and adjacent non-cancerous tissue. Initial analysis with PathSeq comparing viruses with RefSeq genomes revealed that only Adeno-associated viruses (AAV) were differentially and predominantly present in the non-cancer samples (p < 9.4e-6, two-sided Fisher test). However, J. Zucman-Rossi and colleagues (Nault et al., Nat. Genet. 2015; 47:1187-1193) reported that integrations of AAV resembling the strains AAV2 or AAV13 near human genes such as CCNA2, CCNE1 are oncogenic. Understanding when AAV integration is oncogenic is important because AAV2-derived vectors are used in gene therapy. To investigate the apparent discrepancy, we performed AAV-targeted viral capture DNA sequencing of the cancerous tissue and adjacent non-cancerous tissue from each patient with AAV. One patient has an integration of AAV2/13 into CCNA2, expanded in the non-cancer sample and further expanded in the cancer sample that has 10,006 chimeric AAV/CCNA2 DNA reads. We validated that this integration substantially increased mRNA expression of CCNA2, replicating the previous findings. In contrast, we found ten patients with AAV integrations into other loci only in the non-cancer samples and two other patients with AAV integrations into other loci only in the cancer samples. These single-sample AAV integrations did not significantly increase the expression of the target gene, if any, suggesting that they are not oncogenic. All previously reported AAV integrations have been demonstrated only in HCC. Similarly, in our analysis, the only oncogenic integration was in a patient with HCC. Among the ten non-oncogenic integrations in non-cancer samples, one is HCC and nine are iCCA. Both non-oncogenic integrations in cancer samples were in iCCA. Analysis of all previously published AAV integrations and the new AAV integrations we identified showed that almost all the integrations reported as oncogenic overlap the poorly characterized X gene, while the non-oncogenic integrations need not overlap the X gene. This finding explains why the oncogenic AAV integrations are necessarily from AAV2 or a similar strain since other sequenced AAV strains do not contain the X gene. This would suggest re-engineering AAV vectors to make the X gene non-functional, to retain the useful properties of these vectors and concomitantly mitigate the risk of liver cancer in AAV-therapy recipients. Citation Format: Alejandro A. Schaffer, Dana Dominguez, E. M. Gertz, Lesley M. Chapman, Anuradha Budhu, Marshonna Forgues, Jittiporn Chaisaingmongkol, Siritida Rabibhadana, Benjarath Pupacdi, Xiaolin Wu, Curtis C. Harris, Mathuros Ruchirawat, Eytan Ruppin, Xin W. Wang. Oncogenicity of adeno-associated virus (AAV) integration into the genomes of liver cancer patients depends on the overlap between the AAV integration site and the AAV X gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2658.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it’s Insertion into genomic safe harbor AAVS1 site using CRISPR–Cas9

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

Genetic Correction of IL-10RB Deficiency Reconstitutes Anti-Inflammatory Regulation in iPSC-Derived Macrophages

Patient material from rare diseases such as very early-onset inflammatory bowel disease (VEO-IBD) is often limited. The use of patient-derived induced pluripotent stem cells (iPSCs) for disease modeling is a promising approach to investigate disease pathomechanisms and therapeutic strategies. We successfully developed VEO-IBD patient-derived iPSC lines harboring a mutation in the IL-10 receptor β-chain (IL-10RB) associated with defective IL-10 signaling. To characterize the disease phenotype, healthy control and VEO-IBD iPSCs were differentiated into macrophages. IL-10 stimulation induced characteristic signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) downstream signaling and anti-inflammatory regulation of lipopolysaccharide (LPS)-mediated cytokine secretion in healthy control iPSC-derived macrophages. In contrast, IL-10 stimulation of macrophages derived from patient iPSCs did not result in STAT3 phosphorylation and subsequent SOCS3 expression, recapitulating the phenotype of cells from patients with IL-10RB deficiency. In line with this, LPS-induced cytokine secretion (e.g., IL-6 and tumor necrosis factor-α (TNF-α)) could not be downregulated by exogenous IL-10 stimulation in VEO-IBD iPSC-derived macrophages. Correction of the IL-10RB defect via lentiviral gene therapy or genome editing in the adeno-associated virus integration site 1 (AAVS1) safe harbor locus led to reconstitution of the anti-inflammatory response. Corrected cells showed IL-10RB expression, IL-10-inducible phosphorylation of STAT3, and subsequent SOCS3 expression. Furthermore, LPS-mediated TNF-α secretion could be modulated by IL-10 stimulation in gene-edited VEO-IBD iPSC-derived macrophages. Our established disease models provide the opportunity to identify and validate new curative molecular therapies and to investigate phenotypes and consequences of additional individual IL-10 signaling pathway-dependent VEO-IBD mutations.

CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells.

Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.

Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System.

Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. Due to the ease of programming, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-based gene editing tools have gained pace in gene manipulation studies, including investigating complex diseases like cancer. An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn't cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a 'safe-harbor' in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

Efficient viral delivery of Cas9 into human safe harbor

Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nine hemophilia A dogs were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9%−11.3% of normal FVIII levels. In two of nine dogs, FVIII activity increased gradually starting about four years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of these integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A while emphasizing the importance of long-term monitoring for potential genotoxicity.

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in Mice

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population.

Establishment of a type II insulin-like growth factor receptor gene site-integrated SKBR3 cell line using CRISPR/Cas9.

Human epidermal growth factor receptor 2 (HER-2)+ breast cancer has a high recurrence rate and a poor prognosis, with drug resistance contributing to disease progression. The present study aimed to establish a SKBR3 cell line with type II insulin-like growth factor receptor (IGR-IIR) gene site integration using the CRISPR/Cas9 system, and to provide a cell model for exploring the mechanism responsible for the effect of IGF-IIR on trastuzumab resistance in HER-2+ breast cancer cells. In the present study, six single guide (sg)RNA pairs according to the adeno-associated virus integration site 1 (AAVS1) gene sequence were designed and synthesized, and the Universal CRISPR Activity assay CRISPR/Cas9 rapid construction and activity detection kit was used to connect the annealed oligo with the pCS vector. The sgRNA with the highest efficiency was selected to construct a Cas9/sgRNA expression vector using AsiSI + Bstz17I restriction enzymes to cut IGF-IIR. The fragment was ligated into an human AAVS1-KI vector to construct the IGF-IIR targeting vector. The Cas9/sgRNA and IGF-IIR targeting vectors were electroporated into SKBR3 cells, screened using puromycin and identified via PCR, and the mixed cloned cells generated via IGF-IIR gene targeted integration were obtained. The semi-solid and limited dilution methods were used for monoclonal cell preparation, and the results revealed that a Cas9/sgRNA vector that targeted the AAVS1 was successfully constructed. sgRNA activity detection demonstrated that sgRNA2 had the highest efficiency, while enzyme digestion and sequencing confirmed that the IGF-IIR target vector was successfully constructed. The optimum conditions for electrotransfection were 1,200 V, 20 ms and 2 pulses, and the optimal screening concentration of puromycin was 0.5 µg/ml. Using these conditions, the IGF-IIR targeting vector and pCS-sgRNA2 plasmid were successfully transfected into SKBR3 cells, and PCR identification and sequencing verified the correct genotype of mixed clone fragments. The monoclonal cells proliferate slowly and gradually underwent apoptosis. Overall, the present study successfully obtained a mixed clone cell line with site-specific integration of the IGF-IIR gene at the AAVS1.