Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients.Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias. Rearrangements (r) of the KMT2A/MLL gene have been reported in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)-14. AML and ALL patients with KMT2A/MLL-R+ leukemia have a poor prognosis with disappointing event-free survival (EFS) and overall survival (OS) outcomes on contemporary treatment regimens due to relapses caused by cancer drug-resistant clones-19. Therefore, more effective frontline as well as salvage treatments for MLL-R+ acute leukemias are urgently needed. The lysine [K]-methyltransferase 2A (KMT2A)/mixed-lineage leukemia 1 (KMT2A/MLL) gene on chromosome 11 encodes a 431-kDa protein involved in the regulation of transcription+ AML and ALL as potential therapeutic targets to overcome cancer drug resistance. We compared the transcriptomes of primary leukemic cells from KMT2A/MLL-R+vs. KMT2A/MLL-R- AML and ALL patients with an emphasis on the relative gene expression levels of for 21 PTK, including ERBB1, FGR, FLT3, FYN, HCK, JAK2, LCK, LYN, MERTK, SRC, BLK, BMX, BTK, ERBB2, ERBB3, JAK1, JAK3, PTK2, SYK, TEC, TYK2. Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family member TYK2, and the SRC family PTK HCK and FGR were differentially amplified. These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias. The main goal of the present study was to evaluate the protein tyrosine kinase (PTK) profiles of primary leukemic cells in patients with KMT2A/MLL-R. We also used a working database derived from archived datasets including data on primary leukemia cells from 542 adult patients with AML (GSE13159), and 279 pediatric patients with AML  along with the 74 normal/non-leukemic control bone marrow samples (GSE13159) in our comparative gene expression analyses, as described. Probeset level normalization procedures were used as previously reported. A recently published working database, including data on primary leukemia cells from 201 adult patients with B-ALL (GSE13159), 119 pediatric patients with B-ALL (GSE11877 and GSE13351) and 97 infants with B-ALL (GSE68720), as well as 74 normal/non-leukemic control bone marrow samples (GSE13159), was built with previously archived datasets from the NCBI repository and used in our comparative gene expression analyses, as previously described in detail+ pediatric AML patients (N = 48 from GSE17855) with those of primary leukemia cells from 189 FLT3-ITD- pediatric AML patients (GSE17855) as well as normal hematopoietic cells from 74 non-leukemic control bone marrow samples (GSE13159). Forty-seven patients from the KMT2A/MLL-R- “other” group of pediatric AML patients were FLT3-ITD+, including 18 patients with cytogenetically normal AML, one patient with inv AML, 12 patients with t AML, 3 patients with t, 9 patients with AML and other cytogenetic features, and 4 patients who had AML with unknown cytogenetic features. One pediatric patient with KMT2A/MLL-R+ AML was FLT3-ITD+. This double mutant (FLT3-ITD/MLL-R) case was removed from the analysis of FLT3-ITD+  versus KMT2A/MLL-R+ pediatric AML patients . We also compared the gene expression profiles of primary leukemia cells from FLT3-ITD+ AML harbored NPM1 or CEBPA mutations. Within the KMT2A/MLL-R- (“other”) group of KMT2A/MLL-R+ AML pediatric AML patients, 17 harbored NPM1 mutations (NPM1+) and 16 patients had CEBPA mutations (CEBPA+) (GSE17855). We investigated the gene expression profiles from primary leukemia cells from 71 KMT2A/MLL-R- pediatric AML patients who were FLT3-ITD+, NPM1+ or CEBPA+  versus 88 cases of KMT2A/MLL-R+ . One patient with both FLT3-ITD and MLL-R was removed from this comparison.No pediatric patients with KMT2A/MLL-RERBB1, FGR, FLT3, FYN, HCK, JAK2, LCK, LYN, MERTK, SRC, BLK, BMX, BTK, ERBB2, ERBB3, JAK1, JAK3, PTK2, SYK, TEC and TYK2. Standard statistical methods, including mixed model ANOVAs, and hierarchical clustering method were employed, as reported-24. Our analyses for ALL and AML focused on the expression levels (interrogated with 64 probesets) of the following 21 PTK genes: N = 80), children (N = 25) and adults (N = 70) with KMT2A/MLL-R+ B-ALL vs. normal hematopoietic cells from healthy volunteers (N = 74). Notably, FMS-like tyrosine kinase 3(FLT3) expression in infant KMT2A/MLL-R+ ALL cases was 16.09-fold higher than in normal hematopoietic cells in non-leukemic control bone marrow samples  . The genes for several additional PTK showed augmented expression in KMT2A/MLL-R+ infant ALL cells, including TEC, SRC, BLK, JAK2, BTK, PTK (3 probesets), SYK and JAK1 (2 probesets) . As the second most upregulated gene, the gene for BLK was expressed at a 6.77-fold higher level in infant KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells  [Supplementary Table 1]. Similarly, FLT3 wasexpressed at a 22.38-fold higher level in pediatric KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells  . As in infant leukemia cells, BLK was the second most upregulated gene in pediatric KMT2A/MLL-R+ ALL cells showing a 4.38-fold higher expression level than in normal hematopoietic cells . The genes for several additional PTK showed augmented expression in pediatric KMT2A/MLL-R+ ALL cells, including PTK2, TEC, BTK, and SYK . Similarly, FLT3 and BLK expression in leukemic cells from adult patients with KMT2A/MLL-R+ ALL were 21.00-fold  and 21.79-fold , respectively, higher than in normal hematopoietic cells . We first examined PTK gene expression profiles of primary leukemic cells from infants . In infants [Supplementary Table 4], children [Supplementary Table 5] and adults [Supplementary Table 6] with KMT2A/MLL-R+ ALL, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK1, as well as several SRC family PTK, including BLK, were differentially and significantly amplified. In KMT2A/MLL-R+ infant ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to infant ALL cells with germline/wildtype KMT2A/MLL gene [Supplementary Table 4]. Likewise, in KMT2A/MLL-R+ pediatric ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to pediatric ALL cells without KMT2A/MLL rearrangements [Supplementary Table 5]. Similarly, in KMT2A/MLL-R+ adult ALL cells, FLT3_206674_at was the most significantly upregulated probeset  followed by BLK_206255_at  when compared to leukemic cells from adult ALL patients without KMT2A/MLL rearrangements [Supplementary Table 6]. We next compared the PTK gene expression profiles of leukemic cells from each KMT2A/MLL-R+ AML vs. normal hematopoietic cells from healthy volunteers (N = 74). In comparison to normal hematopoietic cells, KMT2A/MLL-R+ pediatric as well as adult AML cells were characterized by amplified expression of FLT3 gene. FLT3_206674_at was the most significantly upregulated probeset  followed by TEC_206301_at  .We examined the PTK gene expression profiles of primary leukemic blasts from 89 children and 38 adults with KMT2A/MLL-R+ AML cells to the PTK gene expression profiles of AML cells without KMT2A/MLL rearrangements from the corresponding control AML patients in the “other” categories . In pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified [Supplementary Table 9]. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified [Supplementary Table 10]. We next compared the PTK gene expression profiles of leukemic cells from pediatric and adult KMT2A/MLL-R+ subset (N = 48) among the pediatric AML patients exhibited upregulated FLT3 expression when compared to non-leukemic control samples (N = 74)  as well as FLT3-ITD- pediatric AML cases (N = 189) . Comparing 189 cases of FLT3-ITD- samples with 48 cases of FLT3-ITD+ pediatric AML samples (GSE17855) exhibited 13 differentially expressed probesets, of which four probesets were significantly upregulated in pediatric FLT3-ITD+ cases. FLT3_206674_at was the most significantly upregulated probeset  followed by BTK_205504_at  and TEC_206301_at  .The FLT3-ITD+ pediatric AML cases with 47 FLT3-ITD+ pediatric AML cases showed 31 differentially expressed probesets, of which 24 probesets were significantly upregulated in pediatric KMT2A/MLL-R+ cases. FGR_208438_s_at was the most significantly upregulated transcript  followed by SYK_207540_s_at  and JAK1_1552611_a_at . ERBB1_1565483_at was the most significantly downregulated transcript in KMT2A/MLL-R+ cases  followed by ERBB1_1565484_x_at  and MERTK_211912_at  A comparison of the 88 KMT2A/MLL-R+ pediatric AML cases with 71 cases of FLT3-ITD+/NPM1+/CEBPA+ pediatric AML cases exhibited 34 differentially expressed probesets, of which 26 probesets were significantly upregulated in the KMT2A/ MLL-R+ subset. FGR_208438_s_at was the most significantly upregulated probeset  followed by SYK_207540_s_at  and HCK_208018_s_at . ERBB1_1565483_at was the most significantly downregulated transcript in KMT2A/MLL-R+ cases  followed by ERBB1_1565484_x_at  and MERTK_211912_at  .A comparison of the 88 KMT2A/MLL-R+ AML and B-ALL patients, especially those with relapsed or refractory leukemia, is disappointingly poor after contemporary treatments-8,10-14. New treatment strategies are urgently needed for both KMT2A/MLL-R+ AML and KMT2A/MLL-R+ ALL. Therefore, a large panel of drugs are being evaluated as potential therapeutic agents against KMT2A/MLL-R+ AML and ALL, including inhibitors of Menin-MLL1 interaction-28. The present study demonstrates that PTK inhibitors (PTKi), especially inhibitors of FLT3, may have clinical impact potential as therapeutic agents against KMT2A/MLL-R+ B-ALL as well as AML. Further, inhibitors of SRC family PTK may be clinically useful against KMT2A/MLL-R+ B-ALL and inhibitors of BTK, SYK, and JAK family PTK may be clinically useful against KMT2A/MLL-R+ AML. If our observations in this small series are confirmed in a larger series of leukemia patients, preferably with corresponding proteomics/phosphoproteomics data, proof-of-concept studies with FDA-approved PTKi would be warranted to further evaluate the clinical potential of PTK targeting in KMT2A/MLL-R+ ALL and AML. The outcome of KMT2A/MLL-R-31. The incorporation of PTKi into the standard of care has caused a paradigm shift in the treatment of CML (PTKi: ABL1 and SRC inhibitors), CLL (PTKi: BTK inhibitors), Ph+ B-ALL (PTKi: ABL1 and SRC inhibitors), and AML with FLT3 mutations (PTKi: FLT3 inhibitors),30. There is growing consensus regarding their evolving role in Ph-like B-ALL  and pre-B ALL with t (PTKi: BTK and SRC inhibitors)-53 [+ B-ALL was associated with a very poor outcome in both children and adults, with less than 20% long-term survival until the introduction of PTKi capable of inhibiting the oncogenic BCR-ABL tyrosine kinase-48 [+ B-ALL are no longer considered to have a poor prognosis. The standard of care is being optimized by developing novel combinations of PTKi such as dasatinib and ponatinib with bispecific antibodies such as blinatumomab, CAR-T cells, and antibody-drug conjugates and using less toxic chemotherapy regimens-48. Likewise, FLT3 inhibitors have contributed to improved outcomes in AML patients with a FLT3 mutation. FLT3 is a receptor tyrosine kinase that plays an important role in normal lymphohematopoiesis. Leukemic cells from AML patients abundantly express FLT3 and many AML patients have activating mutations of FLT3, including internal tandem duplication mutations (FLT3-ITD) and kinase domain activation loop mutations (FLT3-ALM),51. FLT3 mutations are rare in ALL, but a high-level expression of FLT3 was also reported in MLL-AF4 positive ALL patients and showed poor prognostic value,53. Our study significantly expands the knowledge regarding FLT3 expression in ALL as well as AML by demonstrating amplified expression levels in infants, children, and adults with KMT2A/MLL-R+ B-ALL and children with KMT2A/MLL-R+ AML. Several PTKi have been approved for the treatment of AML with FLT3 mutations, including midostaurin and gilteritinib [+ ALL. It is noteworthy that Dovitinib, a multi-functional PTKI with FLT3 inhibitory activity, was reported to exhibit nanomolar in vitro activity against KMT2A/MLL-R+ ALL cells. PTK play a critical role in normal lymphohematopoiesis, and they have also been implicated as leukemogenic oncoproteins in the development of acute and chronic leukemiastors)-53 . Notablyinase-48 . Due to eritinib . Our res+ pediatric AML cells. Except for GSE17855, the archived datasets used in the present study did not contain information about the FLT3 status of the respective leukemia cases. Additional KMT2A/MLL-R+ patients whose leukemia cells do not have amplified FLT3 gene expression but an activating FLT3 mutation like FLT3-ITD may also benefit from the use of FLT3 inhibitors. Analysis of the GSE17855 contributed to our finding that in pediatric patients with KMT2A/MLL-R+ AML, the gene expression level for FLT3 is differentially amplified. It is noteworthy that only one patient with KMT2A/MLL-R+ AML had FLT3-ITD whereas 47 patients from the KMT2A/MLL-R negative “other” group of pediatric AML patients were FLT3-ITD+, including patients with cytogenetically normal AML (N = 18); AML with inv (N = 1); AML with t (N = 12); AML with t (N = 3); AML with other cytogenetic features (N = 9); and AML with unknown cytogenetic features (N = 4). Therefore, our results should not be interpreted to suggest that FLT3 inhibitors would be preferentially active in pediatric AML patients with KMT2A/MLL rearrangements, as many cases without KMT2A/MLL rearrangements whose leukemia cells are FLT3-ITD+ may also benefit from FLT3 inhibitors. Some PTK, such as FLT3, participate in immune suppression mediated by leukemia cells, which may promote the immune escape of leukemic clones. Therefore, their inhibition with PTKi may partially contribute to favorable treatment outcomes by lifting the immune suppression. For example, the PTKi sorafenib has been shown to abrogate the transcriptional downregulation of interferon regulatory factor 7 (IRF7), which resulted in an augmented CD8+CD107a+IFN-γ+ T cell response in mouse models of FLT-ITD+ AML. Mathew et al. proposed that sorafenib may therefore exhibit immune-mediated anti-leukemic activity in FLT3-ITD mutant AML. One of the intriguing findings of our study relates to the amplified expression of the gene for FLT3 in KMT2A/MLL-R-31. FDA-approved SRC kinase inhibitors have become part of the standard of care in the treatment of CML as well as Ph+ ALL-48 [+ B-ALL. The gene for BLK was expressed at a 6.77-fold higher level in infant KMT2A/MLL-R+ ALL cells, 4.38-fold higher level in pediatric KMT2A/MLL-R+ ALL cells, and 21.79-fold higher level in adult KMT2A/MLL-R+ ALL cells than in normal hematopoietic cells. Notably, the expression of the genes for BLK, HCK and LYN in primary leukemic cells from KMT2A/MLL-R+ ALL patients was differentially and significantly amplified regardless of the age group compared to the expression levels in leukemic cells from B-ALL patients without KMT2A/MLL gene rearrangements. Likewise, the genes of several SRC family PTK were differentially upregulated in pediatric and adult KMT2A/MLL-R+ AML cells. The availability of FDA-approved potent inhibitors of SRC family PTK such as ponatinib, bosutinib, and dasatinib [+ leukemias in proof-of-concept clinical trials. The insights and lessons learned in the clinical development of these PTKi as precision medicines against Ph+ ALL should facilitate their development as potential precision medicines against KMT2A/MLL-R+ ALL as well. The SRC kinase family includes several cytoplasmic PTK, including BLK, HCK, FGR, LYN, FYN, and LCK, which have important regulatory functions for signal transduction pathways related to survival, proliferation, and apoptosis of leukemic cells+ ALL-48 . In the asatinib  provides+ acute leukemias. These results extend our earlier studies regarding upregulation of the JAK-STAT pathways in infant B-ALL,57 and upregulation of SYK expression in pediatric B-ALL-62. The availability of FDA-approved potent inhibitors of SYK and JAK provides the opportunity to evaluate their clinical efficacy in KMT2A/MLL-R+ acute leukemias with overexpression of their respective targets. A significant portion of high-risk acute leukemia patients, especially those with KMT2A/MLL-R+ ALL or AML relapse after being treated on contemporary chemotherapy protocols due to cancer drug resistance of their leukemic clones, and the survival outcome of available salvage regimens is disappointing due to the short nature and poor quality of second remissions. We previously reported that the JAK/STAT signaling pathway is constitutively active in infant pro-B ALL cells and treatment with a JAK3 inhibitor or a pan-JAK kinase inhibitor effectively triggered their apoptosis,57. JAK targeting with a small molecule inhibitor may be a viable strategy to overcome cancer drug resistance in KMT2A/MLL-R+ B-ALL cells. Constitutively active JAK2-STAT5 signaling has been shown to be associated with increased surface PD-L1 expression due to amplified PD-L1 promoter activity in myeloproliferative neoplasms, which may facilitate PD-L1-mediated immune escape,64. JAK2 inhibition reduces the PD-L1 expression levels and may therefore mitigate this risk. Additional insights from the present study relate to the upregulation of SYK and JAK expression in pediatric and adult KMT2A/MLL-R-62. Our earlier studies have identified high-level SYK expression as a likely contributor to cancer drug resistance and relapse in B-ALL,61. Upregulation of SYK expression was associated with significant upregulation of at least one of the STAT3 target genes. Inhibition of SYK caused apoptotic death in primary leukemia cells from B-ALL patients that are resistant to chemotherapy. Notably, a nanomedicine candidate containing the SYK-inhibiting small molecule compound 1,4-bis(9-O-dihydroquinidyl) phthalazine/hydroquinidine1,4-phathalazinediyldiether (C61) was capable of destroying > 99.9% of clonogenic B-ALL cells in vivo and thereby improved the event-free survival outcome of SCID mice challenged with otherwise invariably fatal doses of human leukemic B-cell precursors in each of three different xenograft models of chemotherapy-resistant human B-ALL. Taken together with these earlier observations, the amplified expression of SYK in the poor prognosis KMT2A/MLL-R+ ALL patients prompts the hypothesis that SYKi may help overcome the cancer drug resistance of relapse clones and thereby provide the foundation for more effective multi-modality treatment regimens for KMT2A/MLL-R+ acute leukemias.SYK has been discovered to regulate the cancer drug resistance-related anti-apoptotic STAT3, NF-κB as well as PI-3K-AKT-mTOR pathways


Gene therapy for the treatment of malignancies is an emerging and promising area of particular interest. Therefore, it is imperative to develop effective delivery systems for the specific transfer of nucleic acids into tumors. In comparison to viral vectors, non-viral delivery systems tend to be simple to manufacture, show lower immunogenicity, and are associated with fewer regulatory issues when translated into clinical settings. The aim of our study was to develop single-chain antibody conjugated cyclodextrin-modified poly(propylene imine) (PPI) nanocarriers, comprising β-cyclodextrin-modified PPI, mono-biotinylated, maltose-modified PPI, neutravidin and mono-biotinylated prostate stem cell antigen (PSCA)-specific single-chain antibodies for the targeted transposition of minicircle DNA into PSCA-positive tumor cells. Remarkably, we achieved long-term expression of a therapeutic p53 gene in PSCA-positive tumor cells by combining our tumor-specific hybrid polyplexes with the Sleeping Beauty transposon system in minicircle format.p53−/−/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116 Gene therapy for the treatment of malignancies remains an area of particular interest. Previous attempts to deliver gene-coding DNA using replication-deficient viral vectors showed only limited success in preclinical and clinical studies, and have also raised serious safety concerns ,2. In coAmong non-viral gene transfer systems, poly(propylene imine) (PPI) and its surface-modified derivatives have emerged as promising DNA carriers. The high rate of positively charged amino groups on the surface of the PPI dendrimers enables electrostatic interaction with the negatively charged DNA  and resuWe sought to exploit this polyplex system for targeted delivery of gene-encoding therapeutic DNA. As a target on cancer cells, we chose the prostate stem cell antigen (PSCA), a glycophosphatidylinositol (GPI)-anchored tumor-associated antigen which is found in a variety of urogenital-related cancers ,11,12,13To achieve sustained transgene expression of the transgene, we flanked its expression cassette with Sleeping Beauty (SB) inverted terminal repeats (ITRs); these SB transposons are mobile genetic elements that efficiently integrate DNA into the genome. To enable transposition, we employed simultaneous delivery of DNA encoding a hyperactive form of SB transposase (SB100X). In targeted cells, the transposase recognizes the ITRs of the transposon and enables genomic integration via a cut-and-paste mechanism ,21.A major concern when considering the clinical application of gene-coding DNA delivery is the non-specific cytotoxic effects on cells and enhanced degradation of introduced foreign DNA caused by unmethylated CG dinucleotides located in the bacterial backbone of plasmid DNA . To addrp53−/−/PSCA colon cancer cells and PTEN-deficient H4PSCA glioma cells led to decreased clonogenic survival. Remarkably, surviving glioma clones failed to proliferate further. Interestingly, a notable number of colon cancer clones that escaped transgenic p53 showed loss of transgene expression. Noteworthily, in such colon cancer cells, transgenic p53 expression could be induced by treatment with the DNA-damaging antibiotic zeocin. In summary, our results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition for gene therapy, which due to the modular design can be extended to other target genes and tumor entities.To this end, PSCA-specific hybrid polyplexes efficiently delivered MC encoding a GFP transposon in PSCA-positive 293T cells, whereas control hybrid polyplexes conjugated to non-specific control scFv-P-BAPs showed low gene transfer efficiency. In an experimental gene therapy approach, delivery of MC encoding a TP53 transposon by PSCA-specific hybrid polyplexes into p53-deficient HCT1162H and BOP were dissolved in 2 mL of degassed anhydrous DMSO. Subsequently, the resulting reaction mixture was stirred for 2 h. PPI dendrimer solution was slowly added to the activated βCD-PEG-CO2H solution, stirred for 2–2.5 days at room temperature, and dialyzed for 2 days in 5 L water (membrane tube with MWCO 2000 g/mol) with exchanging aqueous solution. After the freeze-drying, a viscous liquid was obtained. Used quantities for CD-PPI (G2) and CD-PPI (G4) synthesis are presented in 1H NMR characterization are presented in 2H.Synthesis of 4th generation maltose-modified PPI dendrimers  14,900 g/mol) and biotinylated mal19-PPIs , 15,490 g/mol) were described previously ,8. For sp53−/− , the near triploid (+/−3n) glioma cell line H4, and the human embryonic kidney cell lines 293T, 293TPSCA, and 293ThuBirA have been described previously  and analyzed under UV light (G:Box Chemi XX9).MC-DNA (1 µg) was incubated for 30 min at room temperature with increasing amounts of PPI or CD-PPI corresponding to mass ratios of 1:5 to 1:0.2. The dendriplexes were then separated by agarose gel electrophoresis  and analyzed under UV light .Analysis of transposon integration was performed by polymerase chain reaction (PCR) using PhusionTM High-Fidelity DNA Polymerase (Thermo Fisher Scientific). 1 × 104 HCT116p53−/−PSCA or H4PSCA cells plated in 6-well plates in DMEM complete were incubated with scFv(AM1)-P-BAP MC-SB100/MC-p53-puroR hybrid polyplexes. Cells treated with scFv(AM1)-P-BAP MC-SB100/MC-puroR hybrid polyplexes were included as mock control. Treated cells were continuously grown in a medium containing 2 µg/mL puromycin starting 24 h after transfection. Clonogenic survival was analyzed after 14 days. In order to investigate the long-term replicative potential of the treated cells, parallel experiments were performed and surviving HCT116p53−/−PSCA and H4PSCA cell clones were pooled. 1 × 103 cells thereof were plated on 10 cm dishes and analyzed after 14 days or grown until 80% confluency for Western blot analysis of transgenic p53 (see above). In all clonogenic survival experiments, cell clones were stained with crystal violet staining solution (Merck Millipore), photographed and counted using ImageJ software .For analysis of direct effects after targeted p53 transposition, 2 × 105 cells were stained with 5 μg scFv-P-BAP for 1 h at 4 °C, followed by secondary anti-biotin-VioBlue antibody . Cells stained with the secondary antibody alone served as the control. To evaluate the targeted delivery of DNA, 2 × 105 293TPSCA cells were incubated with scFv-P-BAP hybrid polyplexes loaded with Cy3-labelled plasmid DNA  for 4 h at 37 °C. Cy3-plasmid-loaded hybrid polyplexes conjugated with control scFv(MR1.1)-P-BAP were included as a negative control. Subsequently, the cells were washed with 0.1% Heparin/PBS (Sigma-Aldrich). To evaluate the transfection efficiency, 1 × 105 293TPSCA were incubated with PPI:MC-GFP dendriplexes containing 1 µg MC-DNA and PPI at different mass ratios for 48 h. At least 2 × 104 cells were measured by MACSQuant Analyser 10 flow cytometer (Miltenyi Biotec) and analyzed by FlowJo software version 10.1 .Binding of biotinylated scFv(AM1)-P-BAP to PSCA-positive target cells and isogenic parental cells was analyzed by flow cytometry. 2 × 105 293TPSCA cells grown on a coverslip were incubated with scFv(AM1)-P-BAP hybrid polyplexes loaded with Cy3-labelled plasmid DNA at 37 °C. After 24 h, cells were fixated with 4% paraformaldehyde in PBS (w/v). Subsequently, cell membranes and nuclei were stained with Alexa Fluor 647-conjugated wheat germ agglutinin  and Hoechst 33,342 (Invitrogen). The early endosomes were stained with EEA1 (E-8) Alexa Fluor 647  according to the manufacturer’s protocols. The coverslips were placed upside down in a mounting medium  on a microscope slide. Cells treated with Cy3-labelled hybrid polyplexes conjugated with non-binding control scFv(MR1.1)-P-BAP were included as negative control. Fluorescence microscopy images were captured by a confocal laser scanning microscope  and analyzed by Fiji software .For visualization of cellular DNA uptake, 6 × 10t-test. Values of p < 0.05 were considered statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001.All experiments were performed at least two times in at least triplicates. Differences between groups were examined for statistical significance using Student’s The toxicity of cationic PPI dendrimers is a major concern, especially when applying them as DNA carriers for cancer therapy. Therefore, we assessed the cell viabilities of 293T cells incubated with increasing concentrations of 2nd generation PPI (G2), 4th generation PPI (G4), and with the corresponding β-cyclodextrin-modified PPIs (PPI (G2) modified with 25% β-cyclodextrin and PPI (G4) modified with 6% β-cyclodextrin (CD-PPI (G2), CD-PPI (G4)) A. We alsPSCA, HCT116p53−/−PSCA and H4PSCA. Detection of scFv(AM1)-P-BAP via a biotin-specific secondary antibody confirmed the accessibility of the biotin residue under native conditions. In comparison, scFv(AM1)-P-BAP did not bind to PSCA-negative isogenic control cells 293T, HCT116p53−/− and H4. As expected, scFv(MR1.1)-P-BAP, which binds to the neo-epitope of the mutated epidermal growth factor variant III (EGFRvIII) did not bind to PSCA-positive target cells and therefore represented a valid negative control scFv for further experiments.All scFv-P-BAP constructs contained an N-terminal Igκ chain leader sequence for extracellular secretion as well as a C-terminal c-myc-epitope and a 6x histidine (His)-tag for detection and purification, respectively A. In addThe stable conjugation of scFv(AM1)-P-BAP to neutravidin was validated by Western Blot analysis shown in For generation of tumor-specific hybrid polyplexes loaded with gene-coding DNA, neutravidin was conjugated with mono-biotinylated scFv-P-BAP and mono-biotinylated mal19-PPI-biotin in a 1:2:1 molar ratio. To avoid potential biotin-driven agglutination due to undesired cross-linking of the components, the remaining biotin-binding pockets of neutravidin were saturated by the addition of D-biotin after conjugation of scFv-P-BAPs and mal19-PPI-biotin. Complexation between CD-PPI (G2) or (G4) and MC was achieved at a mass ratio of 5:1. The electrostatic interactions between the intermediate conjugates mal19-PPI-biotin/neutravidin/scFv-P-BAP (bioconjugation adduct (1) and CD-PPI/MC (bioconjugation adduct (2) resulted in scFv(AM1)-P-BAP-guided hybrid polyplexes B. AssessPSCA were treated with MC-GFP-loaded scFv(AM1)-P-BAP-guided hybrid polyplexes. As negative control, non-specific hybrid polyplexes containing the EGFRvIII-specific scFv(MR1.1)-P-BAP were included. Up to 87.2 ± 2.8% and 64.5 ± 4.7% 293TPSCA cells appeared GFP-positive after treatment with scFv(AM1)-guided hybrid polyplexes containing CD-PPI (G2) and (G4), respectively. Remarkably, transfection efficiencies decreased to 38.6 ± 4.9% and 10% ± 0.9 for hybrid polyplexes containing CD-PPI (G2) or CD-PPI (G4) after conjugation of the control antibody scFv(MR1.1)-P-BAP -P-BAP D,E. Thes1)-P-BAP .PSCA cells. To this end, 293TPSCA were treated with scFv(AM1)-P-BAP-guided hybrid polyplexes loaded with Cy3-labelled plasmid DNA. As negative control, non-specific hybrid polyplexes containing the EGFRvIII-specific scFv(MR1.1)-P-BAP were included. Flow cytometry analysis demonstrated scFv(AM1)-P-BAP-mediated internalization of the hybrid polyplexes by 293TPSCA cells, whereas significantly less Cy3 signals were detectable in cells after treatment with non-specific hybrid polyplexes conjugated with scFv(MR1.1)-P-BAP  demonstrated partial location in early endosomes. In contrast, the non-specific scFv(MR1.1)-P-BAP-guided hybrid polyplexes showed less nanoparticle uptake. Quantitative analysis of Cy3 signals revealed significantly more signals overall and per cell after treatment with scFv(AM1)-P-BAP hybrid polyplexes compared to control hybrid polyplexes -guided hybrid polyplexes containing CD-PPI (G4), which confers most specific transfection efficiencies in 293T1)-P-BAP A,C. Conflyplexes E,F.PSCA cells when transfected with scFv(AM1)-P-BAP hybrid polyplexes loaded with MC encoding GFP. None of the used endocytosis inhibitors completely blocked transfection of GFP, suggesting that besides clathrin-dependent and -independent endocytic pathways a direct uptake mechanisms of scFv-P-BAP-guided hybrid polyplexes might have supported the delivery of transposon MCs encoding GFP . As expected, control cells transfected with only MC encoding transposon-GFP lost GFP expression over time whereas cells transfected with transposon and SB100X showed enduring gene expression in approximately 50% of cells. . In linep53−/−/PSCA and H4PSCA tumor cells. To this end, cells were treated with MC encoding p53-puroR transposon and MC encoding SB. Cells transfected with scFv(AM1)-P-BAP-guided hybrid polyplexes loaded with MC encoding transposon-puroR and MC-SB served as negative control. Whereas the hypodiploid colorectal carcinoma cell line HCT116p53−/− is derived from the parental chromosomally stable cell line HCT116, the near triploid glioma cell line H4 display an increased chromosomal instability [p53−/− cells since continuous mitotic defects and DNA-damage should result in posttranslational modifications  and stabilization of transgenic p53. Analysis of direct effects 10 days after targeted transposition of TP53 revealed decreased clonogenic survival of HCT116p53−/−/PSCA transfected with p53-transposon when compared to mock controls. Yet, the effect of targeted p53 transposition was even more increased in transfected H4PSCA glioma cells when compared to the control (p53−/−/PSCA cell clones that survived scFv(AM1)-P-BAP-guided transposition of p53 were readily propagated onto new culture dishes . Furthermore, expression of p53’s downstream transcriptional target p21waf/cip was noted . DespitRecently, we established a novel tumor-specific siRNA delivery system, consisting of maltose-modified PPI/PPI-biotin, neutravidin, and a biotinylated scFv for targeted siRNA delivery to tumor cells . Yet, sup53−/−/PSCA colorectal cancer cells and chromosomally instable H4PSCA glioma cells demonstrated significant decreased cell survival in response to targeted transposition of TP53 in vitro using our PSCA-specific hybrid polyplexes. While the small amount of surviving glioma cells clones could not be propagated further in cell culture, a notable fraction of colon cancer clones that escaped transgenic p53 showed loss of p53 protein expression and was further passaged in cell culture. Remarkably, in these cells, profound transgenic p53 expression was restored by treatment with the DNA-damaging bleomycin family antibiotic zeocin, which demonstrates efficiency of the transposon system and furthermore suggests that cancer cells with stable karyotype are also a legitimate target for p53 gene therapy.In our concept of hybrid polyplexes for targeted gene therapy, we furthermore sought to avoid delivery of bacterial non-methylated CpG-motifs and to simultaneously implement a transposable element for stable expression of the delivered transgene. This combination should prevent unwanted activation of Toll-like receptor 9 or other pattern recognition receptors in treated cells, eventually leading to inflammation and activation of defense mechanisms, which ultimately lead to degradation of foreign DNA . In thisIn summary, we successfully developed a modular platform technology for the targeted delivery of therapeutic gene-coding DNA to tumor cells. By combining our tumor-specific hybrid polyplexes with the Sleeping Beauty transposon system in minicircle format, we achieved long-term transgenic p53 gene replacement in PSCA-positive tumor cells. In further experiments, the reproducibility for other gene therapy targets needs and feasibility in pre-clinical in vivo models must be investigated to pave the way for potential clinical applications.W.J., S.T., D.A. and A.T. hold IP rights for the hybrid-polyplex system for targeted delivery of DNA to eukaryotic cells.


COVID-19 infection treatment (CIT) has reframed optimizing medication reconciliation in older adults. In December 2021, FDA authorized Emergency Use Authorization of Paxlovid (Nirmatrelvir/Ritonavir), protease inhibitor blocking SARS-CoV-2 replication, to reduce severe COVID-19 and mortality in high-risk patients1. Pharmacodynamic and pharmacokinetic changes in geriatric population warrant a stepwise, patient-centered approach for prescribing Paxlovid by reviewing - 1) drug-drug interactions with current medications2, 2) renal dosing, 3) risks-benefits-alternatives, 4) adverse drug events (ADE), i.e. polypharmacy and prescribing cascade. Case A – Community-dwelling 65-year-old Hispanic male history of paroxysmal ventricular tachycardia and end-stage renal disease (ESRD) requiring CIT- 1) Paxlovid contraindication with Amiodarone2 2) ESRD – not candidate 3) risks outweigh benefits, 4) safer alternatives with less ADE. Case B – Long term care 80-year-old Hispanic female history of stroke, type 2 diabetes mellitus and dementia requiring CIT -1) Paxlovid contraindication with Atorvastatin2 (Atorvastatin held during Paxlovid treatment then restarted at lower dose “deprescribing”), 2) estimated glomerular filtration rate 46 with renal dosing – Paxlovid 150mg/100mg, 3) benefits outweigh risks, 4) monitored ADE in controlled setting. Optimizing medication reconciliation during CIT in older patients can decrease preventable ADE. Further research is essential to align CIT efficacy with the fundamental geriatric principles, including shared decision-making, long-term prognosis, and life care planning. 1Najjar-Debbiny R, Gronich N, Weber G, Khoury J, Amar M, Stein N, Goldstein LH, Saliba W. Effectiveness of Paxlovid in Reducing Severe COVID-19 and Mortality in High Risk Patients. Clin Infect Dis. 2022 Jun 2:ciac443. 2Lexicomp Drug Interaction Checker. Accessed August 6, 2022.