Universal allogeneic CAR T cells engineered with Sleeping Beauty transposons and CRISPR-CAS9 for cancer immunotherapy.

Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an "off-the-shelf" therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR-Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared with the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR-Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.

Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes.

CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Meta-analysis of gene expression profiles identifies differential biomarkers for hepatocellular carcinoma and cholangiocarcinoma.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the members of hepatobiliary diseases. Both types of cancer often exert high levels of similarity in terms of phenotypic characteristics, thus leading to difficulties in HCC and CCA differential diagnoses. In this study, a transcriptome meta-analysis was performed on HCC and CCA microarray data to identify differential transcriptome networks and potential biomarkers for HCC and CCA. Raw data from nine gene expression profiling datasets, consisting of 1,185 samples in total, were methodologically compiled and analyzed. To evaluate differentially expressed (DE) genes in HCC and CCA, the levels of gene expression were compared between cancer and its normal counterparts (i.e., HCC versus normal liver and CCA versus normal bile duct) using t test (P < 0.05) and k-fold validation. A total of 226 DE genes were specific to HCC, 249 DE genes specific to CCA, and 41 DE genes in both HCC and CCA. Gene ontology and pathway enrichment analyses revealed different patterns between functional transcriptome networks of HCC and CCA. Cell cycle and glycolysis/gluconeogenesis pathways were exclusively dysregulated in HCC whereas complement and coagulation cascades as well as glycine, serine, and threonine metabolism were prodominantly differentially expressed in CCA. Our meta-analysis revealed distinct dysregulation in transcriptome networks between HCC and CCA. Certain genes in these networks were discussed in the context of HCC and CCA transition, unique characteristics of HCC and CCA, and their potentials as HCC and CCA differential biomarkers.

Validation of miR-20a as a Tumor Suppressor Gene in Liver Carcinoma Using Hepatocyte-Specific Hyperactive piggyBac Transposons.

We established a semi-high-throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs (miRs) that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding six different miRs from the miR-17-92 cluster and nine miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of oncogenic Harvey rat sarcoma viral oncogene homolog (HRASG12V) and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRASG12V/c-MYC HCC model, consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a overexpression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression.

Transposons: Moving Forward from Preclinical Studies to Clinical Trials.

Transposons have emerged as promising vectors for gene therapy that can potentially overcome some of the limitations of commonly used viral vectors. Transposons stably integrate into the target cell genome, enabling persistent expression of therapeutic genes. Transposons have evolved from being used as basic tools in biomedical research to bona fide therapeutics. Currently, the most promising transposons for gene therapy applications are derived from Sleeping Beauty (SB) or piggyBac (PB). Stable transposition requires co-delivery of the transposon DNA with the corresponding transposase gene, mRNA, or protein. Stable transposition efficiency can be substantially increased by using "next-generation" transposon systems that combine codon-usage optimization with hyper-activating mutations in the SB or PB transposases. By virtue of their relatively large capacity, gene therapy applications with relatively large therapeutic transgenes, such as full-length dystrophin, can now be envisaged. The authors and others have shown that efficient and stable gene transfer can be achieved with these next-generation transposons in several clinically relevant primary cells, such as CD34+ hematopoietic stem/progenitor cells, T cells, and mesenchymal and myogenic stem/progenitor cells that are amenable for ex vivo transfection. Alternatively, in vivo transposon gene delivery has been explored using non-viral vectors or nanoparticles or in combination with viral vectors. The therapeutic potential of these SB- and PB-based transposons has been demonstrated in preclinical models that mimic the cognate human diseases. However, there are still challenges impeding clinical translation of transposons pertaining mainly to the typical limiting efficiencies of most non-viral transfection methods and the intrinsic DNA toxicity. Nevertheless, it is particularly encouraging that transposons have now been used in gene therapy clinical trials. In particular, transposon-engineered T cells expressing chimeric antigen receptors are starting to yield promising results in patients with hematological malignancies.

Preclinical and clinical advances in transposon-based gene therapy.

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.