Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters  for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia. Therapeutic angiogenesis is an approach to restore tissue supply and relieve ischemia by gene delivery in vivo to express angiogenic growth factors (AGF) stimulating blood vessel growth. This strategy has been proposed for treatment of conditions caused by impaired circulation—e.g., chronic lower limb ischemia, diabetic neuropathy, coronary heart disease and myocardial infarction. In recent decades, development of therapeutic angiogenesis using plasmid DNA (pDNA) encoding AGF genes resulted in marketing of gene therapy drugs  and remains an important field in translational research. However, in clinical trials, the majority of non-viral gene therapy drugs encoding vascular endothelial growth factor VEGF165), hepatocyte growth factor (HGF) or basic fibroblast growth factor (bFGF) demonstrated plausible safety profiles with limited efficacy in patients with cardiovascular disease 65, hepat,3.Accumulated data also proposed that delivery of a single AGF is not sufficient to render a long-term positive effect in patients with advanced stages of ischemic disease. Angiogenesis and accompanying nerve growth are multi-staged processes regulated by various cytokines, proteases and growth factors—therefore, a sole factor delivered as a recombinant protein or genetic construct may fail to induce a durable impact. A potential solution is the selection and use of physiologically relevant combinations of factors that may render potential additive effects.Experimental studies in animal models of ischemic disease, including our works, show that delivery of two angiogenic or tissue-protective molecules may substantially enhance efficacy. Combined gene delivery of VEGF165 with angiopoietin-1 , bFGF 5 or HGF ,9 has shOur group has demonstrated the efficacy of intramuscular injection of a mixture of independent plasmids encoding VEGF165 and HGF for stimulation of angiogenesis. Administration of a mixture of pDNAs with VEGF165 and HGF genes to mice with hind limb ischemia resulted in enhanced angio- and arteriogenesis and decreased necrosis in skeletal muscle compared to each factor alone . SimilarDisadvantages of injecting a mixture of vectors include unpredictable probability of co-transfection by several plasmids. It is marginally impossible to predict the rate of transfection by each vector to describe pharmacokinetics and ensure a stable profile of local growth factor concentrations after injection of a mixture of several pDNAs. Furthermore, complex stoichiometry and differences in expression may result in loss of additive effects expected in combined approaches .All this complicates pharmaceutical development of drugs composed by a combination of two or more pDNA constructs. A promising way to overcome these limitations is development of genetic constructs that allow simultaneous delivery of two or more genes via a polycistronic coding vector. General strategies to achieve this goal include potential designs of plasmid DNA with the following elements to provide simultaneous expression of several AGFs see .(1) Vectors with an “internal” promoter where each encoded gene has its independent enhancer, promoter and polyA signal . This de(2) Bidirectional promoters that can be cloned from human and other species’ genomes. These provide transcription of two genes in opposite directions of a DNA strand. A bidirectional promoter must be “strong” and provide high transcriptional activity in different types of cells and must be resistant to silencing by epigenetic factors ,15. Typi(3) Genes can be separated by an internal ribosomal entry site (IRES)—a sequence that ensures binding of ribosome subunits to mRNA and start of protein synthesis without involving the 5′-end of the mRNA. In such constructs, both coding sequences are transcribed under one promoter as a single transcript with genes of interest separated by an IRES. Translation of the gene located upstream of the IRES occurs by a classical cap-dependent mechanism while translation of the second gene is initiated by the IRES in a cap-independent  manner. The most commonly used IRES is cloned from encephalomyocarditis virus (EMCV). The main disadvantage of this design is the reduced expression of the downstream gene which is generally several-fold lower than that of upstream gene ,18,19,20Current study reports on experimental development of a candidate pDNA-based drug to express HGF and VEGF165 in mammalian tissue and evaluation of its therapeutic efficacy in a mouse model of limb ischemia. We believe that the results of this work are of interest for development of novel gene therapy drugs for more efficient treatment of limb ischemia and other cardiovascular diseases associated with impaired blood supply.Codon-optimized complementary DNA (cDNA) of human HGF and VEGF165 was cloned to a nonoptimized first generation DNA vaccine vector (pVAX)-based plasmid.Plasmids carrying different variants of IRES to ensure simultaneous expression of HGF and VEGF165 genes . We used IRES sequences cloned from EMCV, binding immunoglobulin protein (Bip), FGF1 and eukaryotic translation initiation factor 4 G (elF4G) genes see .A vector with the bidirectional cytomegalovirus (CMV) promoter common for the HGF and VEGF165 genes (bi-HGF/VEGF).A vector with two independent promotors: CMV for HGF and CAG for VEGF165 genes (pHGF/VEGF).Three different types of bicistronic vectors were created:Escherichia coli (DH-5α strain) and purified using the EndoFree Plasmid Giga Kit following the manufacturer’s instructions (Qiagen).Plasmid DNA with green fluorescent protein (GFP) gene driven by CMV promoter was used to assure transfection in cell cultures in separate samples. All plasmids were amplified in 2+/phosphate transfection method, which was described earlier -VEGF165 as a basis design, we cloned a panel of candidate pDNAs to screen their expression profiles. Sequences were chosen from published data on IRES efficacy in mammalian tissue ,27EMCV Evaluation of HGF and VEGF165 expression in vitro was performed as described above. Results of the ELISA to evaluate growth factor content in medium collected after transfection are presented in To evaluate angiogenic activity of HGF and VEGF165 produced by HEK293T after transfection by EMCV and Bip, we collected medium samples containing secreted proteins and transferred them to human umbilical vein endothelial cells (HUVECs) on a Matrigel-coated surface.Tube formation after incubation of HUVECs with test samples was evaluated as mean tube length and branching point (BP) number per field of view. As expected, HUVECs showed a comparable angiogenic response after treatment with full endothelial growth medium (EGM-2) serving as a positive control . After HProduction of human growth factors in murine skeletal muscle after pDNA injection was detected by ELISA of medium samples collected from muscle explant cultures at days 3 and 6 ex vivo. AGFs were not detected after injection of EMCV plasmid with EMCV IRES. In medium samples collected from muscle with Bip, IRES plasmid HGF was detected  but VEGF165 amounts were below the limits of detection. Thus, both tested IRES sequences were ineffective for simultaneous delivery of HGF and VEGF165 to mouse skeletal muscle.-pHGF/VEGF—plasmid with CMV and CAG promotors driving HGF and VEGF165 expression, respectively ;-bi-HGF/VEGF—plasmid with bidirectional CMV promotor for both genes.After failure of the IRES-based pDNAs to provide effective production of VEGF165, we switched to a strategy that exploits bicistronic vectors and constructed two new pDNAs A:-pHGF/VHEK293T cells were transfected by pHGF/VEGF or bi-HGF/VEGF and medium samples were collected 48 h after transfection. Using ELISA, we detected both VEGF165 and HGF with a molar concentrations ratio of HGF/VEGF165 of 1.38 for pHGF/VEGF and 2.47 for bi-HGF/VEGF. The highest concentration of both growth factors among all tested vectors was observed after transfection by pHGF/VEGF with independent promotors for genes of interest . Thus, both tested bicistronic vectors showed high expression potency and ratio of HGF to VEGF165 approaching equimolar production of both AGFs.p = 0.00004 and p = 0.006 for “pHGF/VEGF” and “bi-HGF/VEGF”, respectively). Tube formation response was also significantly higher in the “pHGF/VEGF” sample vs. the mock-transfected control .HGF and VEGF165 secreted by HEK293T transfected by pHGF/VEGF and bi-HGF/VEGF were used for tube formation assay in HUVEC culture. Total tube length was significantly higher vs. the DMEM 0.5% FBS control after treatment of HUVECs by a medium from HEK293T transfected by either tested plasmid . The highest caspase-3/7 activity was registered in the mock control and “pHGF/VEGF” samples, while in other tested samples, it was relatively low . The negProduction of human AGF by murine muscle after injection of 100 µg of pDNA was detected by ELISA of medium samples collected from explant cultures at days 3 and 6 of culture. After injection of the bi-HGF/VEGF vector, we detected very high amounts of HGF, but expression of VEGF165 was below the limit of detection. Administration of pHGF/VEGF plasmid resulted in efficient transfection and production of significant amounts of both AGFs. Average concentrations of HGF and VEGF165 at days 3 and 6 after transfection by pHGF/VEGF are presented in Thus, screening of seven plasmid DNA designs has yielded the optimal vector with two independent promoters (pHGF/VEGF) that provided significant amounts of both AGFs in vitro and in skeletal muscle ex vivo. The proportion of HGF/VEGF165 molar concentrations produced by muscle explants transfected by pHGF/VEGF was close to equimolar (≈1–1.2), which allowed for proceeding to animal tests in a hind limb ischemia model to assess its angiogenic efficacy.(1)n = 9;“pHGF/VEGF”—single delivery of pHGF/VEGF (150 μg)*, (2)n = 7;“empty vector”—negative control group with single delivery of empty pDNA backbone  (150 μg)*, (3)n = 8.“saline”—vehicle negative control with 0.9% NaCl injection (150 μL), To evaluate the therapeutic efficacy of pHGF/VEGF on perfusion recovery of ischemic tissue, we utilized a mouse model of hind limb ischemia. After a. femoralis ligation and excision, all animals had a comparable reduction in perfusion rate down to approx. 10% compared to the contralateral limb indicating severe ischemia. No significant deviation of baseline values on day 0 was found among study groups. After induction of unilateral ischemia, mice were distributed to experimental groups and received one of the treatments. Plasmids were delivered in three equal injections (50 μL each) to m. tibialis anterior, m. biceps femoris and m. quadriceps femoris without electroporation. After surgery, all animals received a 1.5-mL bolus of warm sterile saline subcutaneously to compensate for blood loss. The following treatments were used:* All pDNAs were dissolved in 150 μL of saline.p = 0.04). By day 14, these differences increased even more and perfusion in the pHGF/VEGF group was 1.5-fold higher than in the saline group  and 1.33 times higher than in the empty vector . Aggregate data of perfusion dynamics and representative Doppler scans are presented in It is known that C57BL/6 mice have strong vascular repair ability, so for perfusion recovery assessment, we chose the first 14 days after surgery. Limb perfusion measurements by Laser Doppler were performed immediately after ischemia induction (day 0) and at days 7 and 14 after surgery and plasmid injection. We found that single injection of pHGF/VEGF plasmid induced significant restoration of hind limb perfusion at day 7  was significantly higher in the empty vector group compared to pHGF/VEGF and saline negative control: 62.72 ± 5.13% vs. 35.62 ± 4.95% vs. 44.14 ± 6.00%, respectively .Evaluation of angiogenesis was performed by counting CD31+ vessels with and without lumen (capillaries) and α-SMA+ vessels (arterioles) per FOV. A significantly higher number of CD31+ capillaries with lumen was found in pHGF/VEGF group then in saline negative control group: 30.37 ± 4.26 vs. 16.54 ± 2.30 respectively A. A modeDevelopment of effective vectors for combined gene therapy of ischemic disease remains a challenging field, and the lack of prominent candidate drugs clearly slows progress. Our previous studies support the concept of using HGF and VEGF165, known to present a “dynamic duo”, which we characterized as prospective objects for gene delivery. We investigated their pleiotropic effects a well in relevant animal models including limb ischemia and myocardial infarction ,9. Both The present study reports the effort to generate a single-vector non-viral drug for simultaneous delivery of VEGF165 and HGF. In our previous work, using a mixture of pDNAs bearing each factor (VEGF165 or HGF) alone, we found that equimolar production  was a prThe first part of the study clearly demonstrated a lack of expression potency in IRES-based plasmids and that, despite numerous tools that can be used for prediction of vector efficacy experimental, “trial and error” remains the only effective way to yield a candidate drug. IRESs from EMCV, Bip, FGF1 and elF4G genes were tested and demonstrated dramatic differences in protein production in eukaryotic cell cultures depending on the IRES chosen and genes positioned up- or downstream of IRES. Predicting this kind of property for a plasmid vector is marginally impossible, so we believe that our data provide a valid precaution point for developers of similar non-viral delivery systems. Analysis of published data that describe the efficacy of an IRES using reporter genes  or non-mammalian species fails to provide sufficient cues unless you stick to the genes of interest and test the designed vector by all means possible. These results correlate with existing observations—indeed, IRES efficacy will vary depending on the position of gene around the IRES element, species, tissue and cell type and—ultimately—gene of interest ,27.n = 12) with critical limb ischemia [However, a recent pilot study from a competitor group using IRES-based plasmid expressing VEGF165 and HGF reported efficacy of this candidate drug in patients  and bi-HGF/VEGF —showed similar results in human cell culture (HEK293T) with acceptable expression of both AGFs. However, once we compared them “head-to-head” in a mouse explant model, we found that bi-HGF/VEGF completely failed to express VEGF165 yet delivered a very high amount of HGF (>6 ng/mL). Here, one may encounter a species-dependent change in the pDNA expression profile mechanism, which remains enigmatic. It is probable that a bidirectional promoter that was effective in human cells was repressed or lacked a transcription co-activator in mouse skeletal muscle, which resulted in high expression of HGF and lack of VEGF165. This piece of evidence may, on the one hand, question the relevance of mouse-to-human extrapolations, which are formally the basis of most pre-clinical studies. On the other hand, this highlights the importance of maximizing in vitro and ex vivo assessments of “lead” molecules prior to efficacy tests in animal models.In this work, we assessed the effect of HGF and VEGF165 synthesized by HEK293T cells after transfection with experimental plasmids on the survival of HUVECs and their ability to form capillary-like structures. It is known that AGFs promote cell survival, exhibiting anti-apoptotic effects through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and alpha serine/threonine-protein kinase (Akt) pathways . It alsoThe present results show that the efficacy of tube formation in vitro depended heavily on the amount of viable cells, and AGF addition supports cell survival. To study the mechanism, we evaluated the activity of caspase-3/7. In the mock control (medium from HEK293T cells with mock transfection), cell “survival index” was low and activation of caspase-3/7 was observed, which indicates that HEK293T cells might themselves produce proapoptotic factors causing the death of HUVECs. Indeed, transfection-related stress and use of calcium for DNA delivery can induce production of apoptotic bodies and alter transfected cells’ secretome, resulting in a pro-apoptotic spectrum. In the medium collected from HEK293 transfected with the bi-HGF/VEGF plasmid, cell survival was higher than in the mock control, while the fluorescence of the cleaved caspase-3/7 substrate was insignificant, which complies with literature data showing that AGFs have anti-apoptotic properties and can suppress activation of caspase-3/7. Interestingly, in HUVECs treated with a medium from HEK293 transfected with pHGF/VEGF, significant activation of caspases was observed, exceeding that in the mock control, while cell survival was comparable to that in the “bi-HGF/VEGF” sample. It should be noted that a 3-fold higher production of both VEGF165 and HGF was observed after transfection by pHGF/VEGF compared to bi-HGF/VEGF. We can assume that at high concentrations, HGF and/or VEGF165 can stimulate the activation of caspase-3/7 and manifestation of its non-apoptotic functions, which positively affect the survival of endothelial cells and their ability to form capillary-like structures. These findings correlate with data from other authors; pro-apoptotic proteases such as caspase-3, -7, -2, -8 have also been described in non-apoptotic events, such as cell proliferation, differentiation, osteo- and myogenesis, migration, regeneration and cell signaling ,33,34,35Our animal data demonstrated that pHGF/VEGF induced a rapid response to reach significant difference of perfusion vs. saline by day 7 and by day 14; this group achieved superiority over both controls . Empty pDNA is known to induce a certain immune response, especially prominent for vectors enriched by unmethylated cytosine–guanine dinucleotide (CpG) motifs ,37. For Surprisingly, the empty plasmid group showed an increase in ischemic muscle damage and vast necrotic tissue. We believe that this finding should be subject to further investigation as a potential clinical risk to be assessed prior to first-in-human study. We used EndoFree plasmid purification protocols, so we suggest that effects of backbone pDNA are to be evaluated. The vector used in our study was derived from pVAX, known to carry the above-mentioned CpG oligodeoxynucleotides motifs that do stimulate immune cells  and necrAnother important point for this study is that we did not apply any technique to facilitate DNA uptake in the limb ischemia model. Typically, low-voltage pulses, ultrasound or liposomes are used, but we demonstrated that pHGF/VEGF renders a significant impact on limb perfusion even after single “naked DNA” delivery by injection. We believe that in the case of limb ischemia, our approach using multiple-point injections along the limb is a more effective way to facilitate blood vessel growth and remodeling than enhancing transfection efficacy. Our colleagues have shown that multiple intramuscular injections of small amounts of a plasmid solution (muscle chipping) provide plasmid delivery to muscle fibers of equal efficacy to a per saltum injection accompanied by hydrodynamic shock . ClinicaFinally, the angiogenic response profile was notable for the significant difference in an unusual parameter, namely CD31+ blood vessels with lumen. While the other two vessel types—CD31+ capillaries without lumen and α-SMA+ arterioles—failed to reach statistical significance, this may be attributed to the experiment’s duration (14 days) and a clear trend to pHGF/VEGF superiority was obvious.Blood vessels with a CD31+ endothelial cell lining, a lumen and not coated by mural cells (pericytes or smooth muscle cells) may contribute to improvement of skeletal muscle perfusion and their increase may result from cooperative effects of VEGF165 and HGF. This aspect deserves additional discussion and results from existing amplification of VEGF receptors (VEGFR1 and 2) and HGF receptor  signaling cascades in endothelial cells. Once ligands bind their receptors, activation of receptor tyrosine kinases leads to downstream activation of multiple signal molecules  transmitting the signal down to nuclear level to increase endothelial cells’ migration and stimulate proliferation—the cornerstones of angiogenesis. Previous studies have indicated that HGF and VEGF165 may cooperatively activate the ERK1/2 pathway, resulting in increased expression of adhesion molecules (inter-cellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM)) and focal adhesion kinase (FAK) phosphorylation . This isOverall, we can suggest that induction of blood vessel growth driven by pHGF/VEGF has a peculiar feature with formation of small blood vessels with a lumen probably contributing to observed increase of perfusion. We may also assume that by day 14 after injection of experimental pDNA, in most studied specimens, formation of a lumen preceded further taxis of pericytes or smooth muscle cells to complete blood vessel stabilization. The latter is a potent feature of HGF known to attract mural cells to capillaries formed under the influence of VEGFR activation by its ligand ,46.In addition to stimulation of angiogenesis, the combination of HGF and VEGF165 may have a positive pleiotropic influence of peripheral nerve function known to be impaired in limb ischemia and diabetes. Diabetic neuropathy is a painful and hardly curable lesion that emerges from disturbance of vascular and neural trophic, which eventually leads to muscle dystrophy and irreversible frailty. Due to the complementary impact of HGF and VEGF165, we may expect that this condition is a next putative indication for developed pHGF/VEGF. Our previous experience using HGF gene delivery supports its use to stimulate nerve recovery after traumatic injury , while tSupplementation of HGF by VEGF165 is also a reasonable method according to recent clinical data on a candidate drug designated VM202  being developed by Helixmith . VM202 is a plasmid carrying two isoforms of HGF with 728 (HGF728) and 723 (HGF723) amino acid residues intended for the treatment of cardiovascular diseases and painful diabetic neuropathy ,49. In aFrom a clinical point of view, the importance of next-generation gene therapy drugs relying on plasmid DNA can hardly be overestimated. Application of viral vectors for non-hereditary disorders may be limited due to the risk-to-benefit ratio profile. Potential adverse effects and toxicity of adeno-, lentiviruses and adeno-associated viruses (AAV) along with their high cost of manufacture prevent their wide spread in practice . Recent In contrast, plasmid DNAs have shown a favorable safety profile over time, yet efficacy of this approach requires improvement . We beliThe present results indicate that a single administration of pHGF/VEGF induces angiogenesis and may alleviate ischemic necrosis, yet vector effects on inflammatory response are to be investigated in further detail during a pre-clinical study of safety. We also believe that the description of the “pipeline” we used to yield a lead molecule for in vivo tests might be useful for developers of non-viral gene therapy products.Our data indicate that the novel pHGF/VEGF vector is a highly effective and feasible plasmid for therapeutic gene delivery to skeletal muscle. The developed vector provides an attractive option for further development to address unmet medical needs—limb ischemia, peripheral neuropathy and other diseases that can be relieved by effects of HGF and VEGF165 on vascular and neuronal trophic in impaired tissues.


Unravelling the biological processes driving tumour resistance is necessary to support the development of innovative treatment strategies. We report the design and feasibility of the MATCH-R prospective trial led by Gustave Roussy with the primary objective of characterizing the molecular mechanisms of resistance to cancer treatments. The primary clinical endpoints consist of analyzing the type and frequency of molecular alterations in resistant tumours and compare these to samples prior to treatment. Patients experiencing disease progression after an initial partial response or stable disease for at least 24 weeks underwent a tumour biopsy guided by CT or ultrasound. Molecular profiling of tumours was performed using whole exome sequencing, RNA sequencing and panel sequencing. At data cut-off for feasibility analysis, out of 333 inclusions, tumour biopsies were obtained in 303 cases (91%). From these biopsies, 278 (83%) had sufficient quality for analysis by high-throughput next generation sequencing (NGS). All 278 samples underwent targeted NGS, 215 (70.9%) RNA sequencing and 222 (73.2%) whole exome sequencing. In total, 163 tumours were implanted in NOD scid gamma (NSG) or nude mice and 54 patient-derived xenograft (PDX) models were established, with a success rate of 33%. Adverse events secondary to invasive tumour sampling occurred in 24 patients (7.6%). Study recruitment is still ongoing. Systematic molecular profiling of tumours and the development of patient-derived models of acquired resistance to targeted agents and immunotherapy is feasible and can drive the selection of the next therapeutic strategy. In part, this has been feasible due to the improved access of high throughput molecular biology techniques, the improvements in developing patient-derived models and the collaborative efforts of the research community to comprehensively characterize cancer biology3.Cancer research has led to significant advances in the understanding of tumour biology and immunology, providing a rational for the development of novel treatment strategies5. Many innovative approaches, using anti PD1/PDL1 checkpoint inhibitors, kinase inhibitors, antibody-drug conjugates, monoclonal antibodies, cell-cycle inhibitors, endocrine therapies, DNA repair and epigenetic modulators have become standard therapeutic options for selected cancer patients6. This vast landscape of drugs in development, used either as monotherapy or in combination, will continue to improve cancer care in the near future10.In recent years, the breakthrough of highly effective treatments such as immune checkpoint inhibitors and molecular targeted therapies has improved outcomes for patients affected by different types of cancer and radically changed their clinical managementEGFR, BRAF, MET, RET, ROS1, ALK, NTRK, KIT predict responses to specific kinase inhibitors18. PD-L1 staining, tumour mutational burden, T-effector signatures and mutational signatures are currently being studied as predictive biomarkers of treatment with immune checkpoint inhibitors19.In this context, the development of reliable biomarkers is key to predict clinical benefit of therapies and avoid unnecessary toxicities. For instance, targetable molecular alterations in 20. This has subsequently guided the development of novel compounds capable of overcoming these barriers to provide patients with new therapeutic alternatives22.However, even when prolonged disease control can be achieved, disease progression, secondary to acquired resistance to antineoplastic treatments, eventually occur. Multiple resistance mechanisms to targeted therapies have been characterized, shedding light on the evolution of cancer cells under treatment pressure23. Hence, it is crucial to design research strategies intended to systematically study novel resistance mechanisms to cancer therapies.As new treatments are being developed, cancer cells will consequently adapt to sustained tumour proliferation and disseminationHerein, we report the study design and feasibility of the MATCH-R study, a prospective single institution trial, designed to identify mechanisms of acquired resistance in patients with advanced cancer treated with molecular targeted agents and immunotherapy.n = 5), withdrawal of consent (n = 2), absence of tumour biopsy (n = 12) and inadequate tumour content in the biopsy for molecular analysis (n = 11)  Fig. . From thn = 142) followed by prostate (n = 75), urothelial (n = 30), gastrointestinal (n = 17), gynaecological (n = 13) and breast cancers (n = 8). Patients with less frequent tumour types were also included  for the study population was 65 years 55–71) with a higher proportion of men (60.1%)  had experienced disease progression with targeted therapies, 101 (33%) with immunotherapy and 75 (25%) with anti-androgen therapy Fig. .Fig. 2Dr24. Of note, a concomitant double biopsy of progressive and stable lesions were performed in 21 patients. Twelve patients did not undergo tumour biopsy, which was due to technical or clinical factors such as lack of accessible tumour sites, renal insufficiency, previous pneumothorax and anxiety.Overall, the mean tumour content of all 303 biopsies that underwent NGS was 49%. In most cases, the procedure was safe and well tolerated, and procedure-related adverse events were reported in 24 patients among 314 patients who underwent a biopsy (7.6%), of which the most common were the development of pneumothorax (14) and bleeding (2)  Fig. . Of thesn = 59) and castrate-resistant prostate cancer (n = 60) with success rates of 30% and 27%, respectively. Among the 54 established PDX models, 12 were developed from FGFR-driven tumours resistant to erdafitinib, 9 from EGFR-mutant osimertinib resistant tumours and 4 from ALK-rearranged lung cancers resistant to lorlatinib  and quality (percentage of tumour cells) of the PDX samples compared to corresponding patient biopsies.Importantly, when applying a selective pressure using the same drug that the patient had experience before disease progression, 11 out of 12 PDX models tested by XenTech recapitulated the pharmacological response observed in patients, both from progression and stable sites , EGFR (52), FGFR (13) and BRAF (11) Fig. . In addi25. In addition, we described the ROS1 S1986Y/F kinase domain resistance mutations in a patient with ROS1-rearranged lung cancer progressing on crizotinib26. Similarly, multiple oncogenic fusions involving FGFR3, RET or ALK were identified as common resistance mechanisms to the third-generation EGFR TKI osimertinib in EGFR-mutant NSCLC patients27. Lastly, we identified MEK1, NRAS, PTEN and KRAS mutations among BRAFV600E NSCLC patients progressing on dabrafenib-trametinib combination28.In the context of the MATCH-R trial, we have recently reported multiple resistance mechanisms to ALK, ROS1 and EGFR TKIs. For instance, we characterized diverse resistance mechanisms occurring in patients with ALK-rearranged NSCLC progressing on the third-generation ALK inhibitor lorlatinib such as additional ALK secondary compound mutations, and NF2 loss of function mutations35. The BATTLE trial was the first, biopsy mandated, biomarker-based study that demonstrated the feasibility of selecting personalized treatment based on molecular biomarkers revealing an 8-week disease control rate in 46% of chemoresistant NSCLC patients35. In the MOSCATO-01 trial, 33% of heavily pre-treated patients achieved a clinical benefit to targeted therapies, which were assigned based on tumour molecular profiling32. More recently, the WINTHER trial revealed a disease control rate (DCR) of 26.2% when DNA sequencing or RNA expression were used to determine the therapy given to colon, head and neck, and lung cancer patients34. Even more importantly, the I-PREDICT study investigated the feasibility of the selection of a customized multidrug regimen when multiple molecular alterations were identified by DNA sequencing. The authors validated the feasibility of this approach and were able to administer ≥1 matched drug to 49% of patients33. These studies promoted the use of molecular biomarkers to select patients for optimal treatment assignments and revealed that genomic and transcriptomic profiling are both useful for improving personalized cancer treatment recommendations and patient outcome.Systematic molecular profiling of tumours has been proposed as a diagnostic tool to tailor treatment according to the patient’s cancer genotype and phenotype. Previous prospective studies investigated the feasibility of integrating real-time molecular findings for individualized treatmentThe MATCH-R trial is another step forward showing the clinical usefulness of high-throughput genomic and transcriptomic profiling of repeated biopsies in the context of acquired resistance to anticancer agents. Virtually, every patient will eventually acquire a resistance mechanism despite a personalized treatment, and selecting the next treatment regimen in such a context of heterogeneous or adaptive disease remains challenging. The MATCH-R study in addition to providing new insights on acquired resistance mechanisms to a variety of antineoplastic treatments, in a wide range of cancer types, aims at prolonging the patients benefit by readapting the treatment at resistance according to a repeated molecular profiling of patient tumours. Our feasibility results show that 92% of tumour samples with ≥10% tumour cells are suitable for molecular analysis, with complete molecular profiling achievable in 65% of cases. The information obtained from this study is integrated in the clinical context of the patient and discussed in a dedicated molecular tumour board who designs tailored therapeutic options in the setting of resistance. Although the number of patients oriented towards a novel treatment can be limited by drug inaccessibility , 46% (26/57) of patients presenting with an acquired targetable alteration received an adjusted treatment, which is a similar number to what was observed in the I-PREDICT study. Prolonging the clinical benefit for these metastatic cancer patients will remain important and our best option to maximize the clinical outcomes and justifies a costly repeat biopsy program.When feasible, patient-derived in vivo and in vitro models of resistance are developed to further characterize mechanisms of resistance. This collection of PDX/cell line models remains a useful preclinical tool to identify pivotal mechanisms underlying acquired resistance to current targeted therapies and will allow to develop innovative strategies to overcome or prevent treatment failure.We conclude that a systematic and longitudinal study of mechanisms of resistance to targeted therapies and immunotherapy by molecular profiling and the development of patient-derived models is feasible, safe and contributes to clinical benefits for metastatic cancer patients.The MATCH-R trial NCT02517892) is an ongoing prospective, single institution study held at Gustave Roussy Cancer Campus. The primary objective of this study is to characterize molecular mechanisms of acquired resistance to targeted therapies and immunotherapy in patients with advanced cancer by NGS and the development of patient derived xenografts (PDX) and cell lines. Patients must have had either an initial response, defined as partial response (PR) or complete response (CR) by RECIST 1.1, or a stable response (SD) for at least 24 weeks, and develop disease progression while actively receiving molecular targeted therapy or immunotherapy. Key eligibility criteria for study entry are summarized in Table 517892 isBaseline or pre-treatment samples are obtained either from diagnostic formalin fixed paraffin embedded (FFPE) pathology blocks or from fresh biopsies if available. Post-progression tumour samples are obtained by core biopsies stored as frozen samples and embedded in paraffin Fig. , as wellThe expected events for the primary objective are the detection of new molecular alterations, the disappearance of pre-existing alterations, and significant changes in the allele frequencies or the proportion of cells with molecular alterations at the time of resistance. The aim is to identify genes that are altered in more than 10% of the patients who develop resistance to a given drug. Genes for which a pathogenic event is found in at least 2 patients will be selected for further functional laboratory studies, and we plan to study 52 patients per type of molecular targeting agents. With this sample size, the probability to miss a gene that is really altered in more than 10% of the patients who develop resistance is less than 20% (power > 80%).The study was amended from its original design, that required only a post-treatment biopsy (which became cohort 1), to include specific cohorts of patients with paired pre- and post-treatment biopsies (cohorts 2–4). This aimed to increase the precision of this study in the assessment of acquired mechanisms of resistance of anticancer drugs. These cohorts include: patients treated with EGFR/ALK inhibitors in oncogene driven non-small cell lung cancer (NSCLC EGFR+/ALK+) (cohort 2), patients treated with immunotherapy for lung cancer and bladder cancer (cohort 3) and patients with prostate cancer resistant to androgen deprivation therapy (ADT) (cohort 4). Unfortunately the amendment was approved lately and only few patients benefited yet from pre- and post-treatment biopsies (7 in the EGFR/ALK cohort and 13 in the immunotherapy cohort).Patients are included during a consultation with their referring physician and sign the informed consent  sequencer using a customized panel (Mosc4) covering 82 cancer genes developed with Ion AmpliSeq custom design. The bioinformatics analysis is performed using TorrentSuite software, variantCaller (ThermoFisher Scientific). If the proportion of tumour cells is higher than 30%, whole exome sequencing (WES), and RNA sequencing (RNAseq) are also performed in a clinical-grade laboratory. For WES, the mean coverage was 140X.Clinical Actionability of molecular Targets (ESCAT) and the OncoKB databases are used to rank genomic alterations as potential targets for cancer precision medicine and to predict the effects and treatment implications of specific cancer gene alterations. According to the availability of registered drugs or ongoing clinical trials, patients are oriented towards adjusted treatment strategies whenever possible.On a weekly basis a group of 10 people composed of clinicians, biologists and scientists review the sequencing results and generate a report. Targeted NGS results are usually discussed first but WES and RNA sequencing data are also incorporated into the discussions as soon as they are available, in order to confirm or complete the molecular profile. For every patient included in the MATCH-R trial, the ESMO Scale for Fresh tumour fragments are implanted in the sub-renal capsule of NOD scid gamma (NSG) or nude mice obtained from Charles River Laboratories. Xenografts are then serially propagated subcutaneously from mice to mice up to five passages to generate a viable tumour bank. From passage 3, selective pressure with the inhibitor to which the patient acquired resistance is applied to the mice, to avoid expansion of sensitive tumour cell populations. This is performed through a collaboration with the PDX-dedicated CRO (XenTech).TM dissociator. Cells are cultured with DMEM/F-12+GlutamMAXTM 10% FBS and 10% enriched with hydrocortisone 0.4 µg/ml, cholera toxin 8.4 ng/ml, adenine 24 µg/ml and ROCK inhibitor 5 µM  until a stable proliferation of tumour cells is observed.Patient-derived cell lines are developed from (a) patient biopsies or (b) PDX samples. (a) Patient biopsies are cut in petri dishes and incubated with Liberase™ DH Research Grade  at 37 °C for 1 h; (b) PDX samples are processed by enzymatic digestion with the tumour dissociation kit  and mechanic degradation with the gentleMACsMinistère de ‘Enseignement supérieur, de la Recherche et de l’Innovation (APAFIS#2790-2015112015055793).All patients participating in the study are fully informed and sign an informed consent. The MATCH-R trial has been approved by the ethics committee at Institut Gustave Roussy, the French National Agency for Medicines and Health Products Safety (ANSM), and is conducted in accordance with the Declaration of Helsinki. Demographic and clinical data are prospectively collected together with pathology records and integrated with molecular analysis and translational research studies. All animal procedures and studies have been approved by the French Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary


Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5%  to approximately 60%  across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology.Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on  This isde novo synthesis of other metabolites like non-essential amino acids. It has even been hypothesized that the coevolution between mammalian herbivores and their intestinal bacteria was a factor in the Cenozoic mammal radiation [4 grasses evolved and spread throughout the globe [To survive and reproduce on a low-quality diet, most herbivores and omnivores maintain a community of symbiotic bacteria  that canadiation ,8, espeche globe . In addihe globe .The two most dominant bacteria phyla found in the intestines of mammalian herbivores are Firmicutes and Bacteroidetes , and thede novo with carbon from dietary carbohydrates, lipids, or dietary amino acids (AAs) catabolized during metabolism [ESS needs of eukaryotes must be routed directly from dietary protein or supplied by the gut microbiome.Exploring the complex functional role(s) that the gut microbiome plays in the protein metabolism of mammals is a burgeoning field. Eukaryotic hosts can acquire non-essential amino acids in several ways : (i) routabolism –20, and/tabolism ,21,22. BESS and non-essential AAs can be routed by the host (RD pathway) or associated gut bacteria (RM pathway). Third, bacteria can also synthesize de novo all AAs required for protein biosynthesis [de novo synthesis of AAESS by gut bacteria may be sourced from non-protein dietary macromolecules (carbohydrates or lipids) or catabolized dietary protein and thus is potentially important in situations where dietary protein is limited; even some non-essential AAs can be conditionally essential in rapidly growing animals [de novo or directly route them into their cells.The potential sources of AAs and the pathways by which they can be assimilated by the host are numerous . Dietaryynthesis , which i animals . Gut mic animals ,26 and u15NH4Cl) show that the gut microbiome can provision its host to various degrees with AAESS [ESS in the host plasma pool [δ13C) of individual AA at natural abundances, our experiment on an omnivorous fish found that the carbon in the AAESS in the muscle of tilapia (Oreochromis niloticus) fed diets low in protein (less than 4%) was sourced from dietary carbohydrates [ESS in tilapia muscle when fed low-protein diets. A similar approach has been applied to phytophagous insects that persistently consume AA-deficient diets [While the role gut microbiota play in the protein metabolism of wild animals has not been systematically explored, lab experiments utilizing isotopically labelled substrates  protein and C4-based (δ13C: −12.0‰) carbohydrates. We then used 16S rRNA gene analysis of mice ceca, AA δ13C analysis, and a series of models to estimate how the microbial contribution of each of six major AAESS to muscle protein varied across diet treatments. We predicted that the composition of the gut bacterial community would be sensitive to dietary protein: carbohydrate content. We also predicted that microbial contribution of individual AAESS would increase with decreasing dietary protein content. Our novel combination of next-generation genetic sequencing and isotope analysis of a large suite of individual AAESS enabled us to quantify the degree to which the gut microbiome contributes on a compound-specific level to the AAESS pool used by its host to synthesize skeletal muscle.To understand the functional significance of the microorganisms responsible for this important and likely common biochemical pathway in wild omnivorous and herbivorous animals whose growth, overall health, homeostasis, and reproduction are often limited by protein availability, we conducted a controlled feeding experiment with 2.(a)Mus musculus) purchased from Charles River Laboratories  and randomly divided into four treatment groups (n = 10/group) were housed in four plastic cages at 25°C with a 12 L : 12D photoperiod. Each treatment received one of four diets that varied inversely in C3-based protein (δ13C = −26.5‰) versus C4-based carbohydrate content, which included sucrose (δ13C = −12.2‰) that remained in constant proportion (36%) among treatments, and cornmeal (δ13C = −12.0‰) that varied inversely with protein content . Note that the cornmeal used in our experiment was 10% protein by weight. All work was conducted with the approval of the University of New Mexico Institutional Animal Care and Use Committee (#A-4023-01). While lab-reared mice typically host the same suite of microbial phyla in their guts as wild rodents, they support higher relative proportions of Firmicutes to Bacteroidetes [Sixty recently weaned approximately 3- to 4-week-old Swiss Webster house mice (roidetes  and as s(b)SRX6959962 through SRX6959981).Mouse ceca were dissected under sterile conditions from five randomly selected mice from each diet, preserved in equal volumes of sucrose lysis buffer  and stor(c)2 via a Thermo Scientific IsoLink combustion interface and analysed with a Thermo Scientific Delta V Plus isotope ratio mass spectrometer at the University of California Merced .Mouse muscle tissue was lipid-extracted, hydrolysed, derivatized, and corrected for carbon added during derivatization ; additio(d)ESS concentrations to estimate the availability of dietary AAESS sourced from both casein and cornmeal, which was 10% protein by weight. This first model (Model #1) generated treatment-specific δ13C values for AAESS that could be directly routed from diet, which for most diet treatments was overwhelmingly casein but did include a significant cornmeal component in the low-protein diet treatment. Second, we used another linear mixing model to estimate the proportion of each AAESS in mouse muscle that was directly routed from dietary protein versus that sourced from dietary carbohydrates . This second model (Model #2) used the δ13C values generated by the first as one potential source and predicted δ13C values of AAESS synthesized by microbes from dietary carbohydrates that were estimated with AA-specific isotopic fractionation factors reported in [ESSδ13C values of mouse muscle and our two end member sources in a linear mixing model to estimate the proportion of AAESS in mouse muscle that was ultimately derived from microbial synthesis versus directly routed from dietary protein.We applied a linear mixing model that incorporated weight per cent diet proportions and associated AAorted in ,41 as th(e)ESSδ13C was investigated by comparing operational taxonomic unit (OTU)-level Bray–Curtis dissimilarities among samples to AAESSδ13C values using canonical redundancy analysis (RDA). The significance of results was assessed through 1000 permutations.The degree to which microbial community composition impacted host muscle AA(f)ESS and Asp for host metabolism. We also used published estimates of AA requirements for rapidly growing mice [We used a combination of data on dietary protein content and published estimates of digestibility  and mucoing mice ,45 to caing mice ,46; we uing mice  of 30% f3.(a)The rate of mass gain did not vary among diet treatments between Week 4 and Week 15 . We assume that muscle tissue harvested at the end of our feeding experiments (Day 120) was in steady state with diet because of the observed increases in body mass, and the results of previous feeding experiments that estimated carbon isotope incorporation rates for muscle of adult mice , which h(b)a); samples clustered separately by diet  when analysed using Bray–Curtis distances (a). Random Forests, a machine learning algorithm used for classification, could distinguish samples from the diet treatments with 95% accuracy. The ratio of baseline error to observed error was 15; a minimum ratio of 2 is expected for factors that can be accurately predicted [Gut bacterial diversity increased with dietary protein content  and community composition varied among the individual ceca samples by diet treatment a; sampleistances a. Randomredicted .Figure ρ = 0.53, p = 0.017) to diet treatment, with the most Firmicutes found in mice fed low-protein/high-carbohydrate diets (b). Specifically, Firmicutes were most abundant (82.5% and 66.2%) in Diet #3 (12P:55C) and Diet #4 (9P:75C), respectively. By contrast, the abundance of Firmicutes relative to Bacteroidetes was more even in Diets #1 (40P:40C) and #2 (21P:45C) with 49.5 and 45.5% Firmicutes and 46.9 and 47.6% Bacteroidetes, respectively (c). At the family level (d), microbial communities were primarily composed of Lactobacillaceae, Lachnospiraceae, Ruminococcaceae (Firmicutes), Bacteroidaceae, Porphyromonadaceae, Rikenellaceae, S24-7 (Bacteroidetes), Alcaligenaceae (Proteobacteria), Deferribateraceae (Deferribacteres), and Verrucomicrobiaceae (Verrucomicrobia). OTUs from these families contributed most significantly to the classification of samples by Random Forests among the high- and low-protein diets ESSδ13C values of mice muscle, as well as end member values for AAESS from dietary protein and microbial AAESS synthesized de novo with carbon from dietary carbohydrates for the four diet treatments; see electronic supplementary material, table S4 for mean (±SD) δ13C values. In general, mice muscle AAESSδ13C values were more similar to those in dietary protein for the high-protein diets (a) than low-protein diets (b). Most mice muscle AAESS in the high-protein diet treatments (a) had δ13C values that were closer to those of dietary protein (open blue circles), while mice muscle AAESS in the low-protein treatments (b) was more intermediate between dietary protein (open red/orange circles) and AAESS synthesized de novo by gut microbiota from dietary carbohydrates (open grey circles).in diets a than loin diets b. Most meatments a had δ13eatments b was morESS sourced from gut microbiota make an important contribution to those used by mice to synthesize muscle tissue  and #2 (21P:45C) (p > 0.05), but the Diet #3 (12P:55C) model had an R2a value of 0.37 (Akaike Information Criteria (AIC) = 12.2, F-value = 3.37, p = 0.008) and the Diet #4 (9P:75C) model had an R2a value of 0.43 .Mixing models confirmed that AAe tissue . As expe(d)ESS in diet treatments containing ≥12% protein , an important intermediary for the de novo synthesis of Thr and Lys, was much lower than metabolic demand for all diet treatments after accounting for catabolism by intestinal mucosa  and thu content , which c(a)a) and the ratio of Bacteroidetes versus Firmicutes (figure 2b) were strongly influenced by diet. Firmicutes were a greater proportion (approx. 60–98%) of the communities in the mice fed the low-protein (high-carbohydrate) diet, whereas the relative abundance between these two phyla was more even in the high-protein diets.The vertebrate gut microbiome is dominated by two main bacterial phyla, the Firmicutes and Bacteroidetes ,52 and afigure 2c) and played a signifcant role in classifying the gut communities according to diet treatment ESS to host protein metabolism is the balance between the availability of AAESS in dietary protein and the host's metabolic demand of AAESS required for tissue growth and maintenance. Combining the two sources of dietary protein  and comparing their relative supply across diet treatments directly to their demand based on data for rapidly growing mice [ESS we measured in all but the low-protein diet , isotope-based estimates of the gut microbial contribution to mouse muscle synthesis varied from approximately 15% to nearly 40% when mice were fed high-protein diets that far exceeded their requirements for optimal growth  is required to synthesize aspartate semialdehyde, an intermediary in both Thr and Lys synthesis. Dietary supply of Asp was much lower than metabolic demands in all diet treatments [ESS is related to metabolic costs associated with their de novo synthesis by the gut microbiome. There are costs in terms of energy (ATP and NADPH) as well as the number of steps and types of intermediaries involved in specific biochemical pathways needed to synthesize AAESS from carbohydrates [ESS  or oxaloacetate, a precursor that is an intermediary in the tricarboxylic acid (TCA) cycle. Three of the four AAESS  that showed high (15–40%) gut microbiome contributions in mice fed high-protein diets are branch-chained AAs that play vital roles in protein synthesis and catabolism, cell signalling, and the metabolism of glucose [ESS in the other two precursor families [ESS, even when mice were fed high-protein diets. By contrast, the high microbial contributions of Ile are difficult to explain given how metabolically expensive it is to make  relative to Val, and that relative dietary supply of Ile exceeded metabolic demands in all treatments except the diet with the lowest protein content  needed to synthesize it from phosphenolpyruvate, Phe is costly to make in comparison to most of the other AAESS we measured. Thus, it's not surprising that microbial contributions were relatively low in comparison to AAESS that belong to the pyruvate precursor family. Moreover, in contrast with other AAESS, the observed isotopic pattern  is strongly correlated with the composition of the microbiome community. Specifically, mice used a significant proportion of microbially derived AAESS and had bacterial microbiomes dominated by Firmicutes when fed diets low in protein but full of simple carbohydrates (sucrose). Whether Firmicutes are responsible for synthesizing these AAESS is currently unknown, however, the high proportions of microbially derived AAESS observed in mouse muscle shows how important the gut microbiome as a whole is for mediating AA metabolism in the host. Because microbes can affect the isotopic composition of AAs in animal tissues, ecologists who increasingly use the carbon isotope fingerprints of AAs to determine the ultimate sources of primary production to an animal's diet [ESS budget used to synthesize their tissues. As we quantify the role of the gut microbiome in the protein metabolism of more host organisms, the phrase ‘you are what you eat’ becomes more appropriate than previously considered, since our study shows that a combination of both exogenous as well as endogenous sources of nutrition derived from the gut microbiome may influence host fitness.Overall, our data show that the degree of AAl's diet  need to l's diet . Clearly


Plasmodium spp., the molecular players involved in mitochondrial fission and their mechanisms of action remain largely unknown. Here, we found that the only identifiable mitochondrial fission adaptor protein that is evolutionarily conserved in the Apicomplexan phylum, Fis1, it not essential in P. falciparum asexual stages. Our data suggest that malaria parasites use redundant fission adaptor proteins on the mitochondrial outer membrane to mediate the fission process.Malaria is responsible for over 230 million clinical cases and ∼half a million deaths each year. The single mitochondrion of the malaria parasite functions as a metabolic hub throughout the parasite’s developmental cycle (DC) and also as a source of ATP in certain stages. To pass on its essential functions, the parasite’s mitochondrion needs to be properly divided and segregated into all progeny during cell division via a process termed mitochondrial fission. Due to the divergent nature of  Plasmodium spp., the single mitochondrion grows from a small tubular structure to a complex branched network. This branched mitochondrion is divided at the end of schizogony when 8 to 32 daughter cells are produced, distributing one mitochondrion to each forming merozoite. In mosquito and liver stages, the giant mitochondrial network is split into thousands of pieces and daughter mitochondria are segregated into individual progeny. Despite the significance of mitochondrial fission in Plasmodium, the underlying mechanism is largely unknown. Studies of mitochondrial fission in model eukaryotes have revealed that several mitochondrial fission adaptor proteins are involved in recruiting dynamin GTPases to physically split mitochondrial membranes. Apicomplexan parasites, however, share no identifiable homologs of mitochondrial fission adaptor proteins with yeast or humans, except for Fis1. Here, we investigated the localization and essentiality of the Fis1 homolog in Plasmodium falciparum, PfFis1 (PF3D7_1325600), during the asexual life cycle. We found that PfFis1 requires an intact C terminus for mitochondrial localization but is not essential for parasite development or mitochondrial fission. The dispensable role of PfFis1 indicates that Plasmodium contains additional fission adaptor proteins on the mitochondrial outer membrane that could be essential for mitochondrial fission.Malaria remains a huge global health burden, and control of this disease has run into a severe bottleneck. To defeat malaria and reach the goal of eradication, a deep understanding of the parasite biology is urgently needed. The mitochondrion of the malaria parasite is essential throughout the parasite’s life cycle and has been validated as a clinical drug target. In the asexual development of IMPORTANCE Malaria is responsible for over 230 million clinical cases and ∼half a million deaths each year. The single mitochondrion of the malaria parasite functions as a metabolic hub throughout the parasite’s developmental cycle (DC) and also as a source of ATP in certain stages. To pass on its essential functions, the parasite’s mitochondrion needs to be properly divided and segregated into all progeny during cell division via a process termed mitochondrial fission. Due to the divergent nature of Plasmodium spp., the molecular players involved in mitochondrial fission and their mechanisms of action remain largely unknown. Here, we found that the only identifiable mitochondrial fission adaptor protein that is evolutionarily conserved in the Apicomplexan phylum, Fis1, it not essential in P. falciparum asexual stages. Our data suggest that malaria parasites use redundant fission adaptor proteins on the mitochondrial outer membrane to mediate the fission process. Plasmodium parasites inside the red blood cells (RBCs) and periodic rupture of the infected RBCs. After invasion of a host cell, the parasite undergoes growth and division within the parasitophorous vacuole to generate new infective progeny. Unlike many cells that divide via binary fission, the malaria parasite replicates its nuclear DNA for 3 to 5 rounds without concurrent cytokinesis, resulting in the formation of 8 to 32 daughter cells (merozoites). This unique mechanism of division starts at the very end of the asexual life cycle and is termed schizogony   (www.PlasmoDB.org) did not find any homologs of other mitochondrial fission adaptor proteins in humans or yeast . In addition, Fis1 is extremely highly conserved across various Plasmodium species (www.PlasmoDB.org). Overall, Fis1 seems to the only evolutionarily conserved mitochondrial fission adaptor protein present throughout eukaryotic kingdoms.Studies on mitochondrial fission in model organisms have suggested that the fission machinery that “splits” mitochondrial membranes involves at least two classes of molecules: mitochondrial fission adaptor proteins located on the mitochondrial outer membrane (MOM) and fission GTPases (mechanoenzymes) that are recruited from the cytosol to the mitochondrial fission sites . In mammevisiae) , plants evisiae) , or unicevisiae) . In apicevisiae) ; BLAST sP. falciparum  shares 30% sequence identity with human Fis1. A modeled three-dimensional (3D) structure of PfFis1 generated with I-Tasser software (1PC2) (data not shown). In asexual blood stages, PfFis1 is transcribed at all stages, with peak transcription in the late trophozoite and early schizont stages  study carried out in this model species (https://plasmogem.sanger.ac.uk/)   for anchoring in the MOM and a short C-terminal sequence (CTS), whereas the N terminus has two tetratricopeptide repeats (TPR1/TPR2) that facilitate protein-protein interactions . The hypsoftware  is also t stages , which iIn this study, we generated a conditional PfFis1 knockdown (KD) line and a PfFis1 KO line via CRISPR/Cas9-mediated genome editing. Primers used to perform these genetic studies are listed in P. falciparum, we episomally expressed tagged PfFis1 fusion proteins with small epitopes in D10 wild-type (WT) parasites. In one transgenic line, we tagged PfFis1 with 3× hemagglutinin (3HA) at the N terminus (www.PlasmoDB.org). Expression of tagged PfFis1 at the predicted molecular weight was confirmed by Western blotting  resulted in mislocalization of the protein to the cytoplasm  with the template plasmid, pMG75noP-Fis1-8apt, and with two guide RNA (gRNA) constructs that were designed to guide Cas9 cleavage near the end of the PfFis1 locus. The transfected parasites were selected using media containing blasticidin and anhydrotetracycline (aTc), yielding a conditional KD line. The small molecule aTc maintains the expression level of the target gene by preventing the negative regulator, the TetR-DOZI fusion protein, from binding to the target mRNA via RNA aptamers  are homologous to the ends of the pLN-hDHFR-3HA construct present after restriction digestion with AvrII and AflII sites . The seqhttp://grna.ctegd.uga.edu/). They were individually cloned into the NFCas9-yDHOD(-) plasmid, which contains a full-length Cas9 gene from Streptococcus pyogenes without any tags and a gRNA expression cassette  were identified from the gene sequence using the Eukaryotic Pathogen CRISPR guide RNA design tool  of PfFis1 was amplified from genomic DNA by the use of primers P13 and P14. The 3′UTR (3′ homologous region) of PfFis1 was amplified from genomic DNA by the use of primers P15 and P16. The 3′UTR was cloned into the pMG75noP-8apt-3HA vector  using AfThese procedures followed our published protocols , 21.−ΔΔCT) method as previously described (Taq polymerase was kept at 62°C.Genomic DNA from late-stage parasites was isolated with a DNeasy blood and tissue kit (Qiagen). During the KD time course , total RNA from parasites representing each set of conditions (aTc plus versus aTc minus) was isolated from saponin-lysed parasite pellets followed by treatment with TRIzol (Thermo) and purification with an RNeasy kit (Qiagen). After treatment with DNase I (New England Biolabs), 2 μg RNA from each set of conditions was primed with random hexamers and converted to cDNA using SuperScript III reverse transcriptase (Thermo). qRT-PCR was carried out in triplicate with SYBR green real-time PCR master mixes (Thermo) in a real-time PCR instrument (Applied Biosystems). Primers used for amplification of PfFis1 are listed as P30 to P31. A previously reported housekeeping gene, encoding seryl-tRNA synthetase, was used as the internal control (primers P32 to P33) . Data weescribed . For reg


Using immunohistochemistry, enterovirus capsid proteins were demonstrated in pancreatic islets of patients with type 1 diabetes. Virus proteins are mainly located in beta cells, supporting the hypothesis that enterovirus infections may contribute to the pathogenesis of type 1 diabetes. In samples of pancreatic tissue, enterovirus RNA was also detected, but in extremely small quantities and in a smaller proportion of cases compared to the enteroviral protein. Difficulties in detecting viral RNA could be due to the very small number of infected cells, the possible activity of PCR inhibitors, and the presence—during persistent infection—of the viral genome in unencapsidated forms. The aim of this study was twofold: (a) to examine if enzymes or other compounds in pancreatic tissue could affect the molecular detection of encapsidated vs. unencapsidated enterovirus forms, and (b) to compare the sensitivity of RT-PCR methods used in different laboratories. Dilutions of encapsidated and unencapsidated virus were spiked into human pancreas homogenate and analyzed by RT-PCR. Incubation of pancreatic homogenate on wet ice for 20 h did not influence the detection of encapsidated virus. In contrast, a 15-min incubation on wet ice dramatically reduced detection of unencapsidated forms of virus. PCR inhibitors could not be found in pancreatic extract. The results show that components in the pancreas homogenate may selectively affect the detection of unencapsidated forms of enterovirus. This may lead to difficulties in diagnosing persisting enterovirus infection in the pancreas of patients with type 1 diabetes. Enteroviruses (EVs) are linked to the pathogenesis of type 1 diabetes (T1D). Epidemiological studies showed a risk association between EV infections and the development of islet autoimmunity and T1D, and recent reports indicated that a low-level EV infection may occur in the pancreata of patients with recent onset T1D ,3,4, wheIn fact, detection of EV RNA in the pancreas of T1D patients turned out to be challenging, and the amount of genome equivalents is extremely low and close to the detection limits of sensitive RT-PCR assays . One reaThus, there is an intense interest in whether persistent infections as reported above can also be found in pancreata of T1D patients. In addition to the supposedly low virus amount, the pancreas may also contain compounds that inhibit PCR reactions. The above observations may explain why detection of EV RNA in the pancreas of T1D patients is so difficult. The nPOD (Network for Pancreatic Organ Donors with Diabetes) pancreas tissue biobank  greatly We set out to evaluate the sensitivity of EV detection using different RT-PCR methods in pancreas homogenates. Particularly, we addressed the question whether pancreatic components may affect the ability of RT-PCR methods to detect encapsidated vs. unencapsidated virus RNA including the terminally deleted form of viral RNA. We also addressed if the organ procurement procedure (including transport to the laboratory on wet ice) could affect virus detection. This study was performed at three collaborating laboratories of the nPOD-V working group. Thus, we could compare the sensitivity of EV detection using five different RT-PCR approaches targeting the conserved 5′ UTR region of viral genome.Three virology laboratories of the nPOD-V consortium contributed to the study: (a) University of Tampere, Finland; (b) Baylor College of Medicine, Houston, Texas; (c) University of Insubria, Varese, Italy.−3, 10−6, 10−7, 10−8, 10−9) and immediately frozen at −80 °C. One set of CVB1 dilutions was kept on wet ice for 20 h after spiking and frozen as above. An identical CVB1 dilution series was made in sterile water. Pancreas extract and water samples with no addition of virus were used as negative controls.An EV-negative pancreas sample of a non-diabetic organ donor from the PanFin study  was homoFrozen samples were coded and distributed to the three laboratories. Blinded samples were tested at the three nPOD-V laboratories that used five different RT-PCR methods for EV detection. Tampere laboratory: RNA was extracted from pancreas homogenate samples using the Viral RNA Kit . Samples were analyzed using two RT-PCR methods: a semi-quantitative RT-PCR coupled with hybridization with an EV-specific probe (PCR 1) [ (PCR 1) , and a q (PCR 1) .Houston laboratory: RNA was extracted with MagMax Viral RNA Isolation Kit . RNA was converted to complementary DNA (cDNA) with Superscript III RT (Invitrogen) according to the manufacturer’s directions with random primers. PCR was carried out with SYBR-Green PCR master mix (Invitrogen) using the same primers as above [as above . PCR wasVarese laboratory: Pancreas homogenates were analyzed using two different procedures: (a) direct RNA extraction (PCR 1), and (b) RNA extraction following blinded passage in four different cell lines  in order to enrich the possibly present virus (PCR 2). The procedure was performed essentially as reported [reported . Total RAll used enterovirus primer pairs and probes are listed in 5 cells per well. Cells were transfected with a combination of the CVB3 RNA and plasmid DNA expressing GFP (green fluorescent protein) which served as carrier nucleic acid and as a marker of transfection efficiency. For each well, a combination of GFP plasmid DNA (250 ng) and 1.2 × 106 genome copies of either CVB3 RNA or TD-CVB3 RNA was transfected using Lipofectamine 3000 (Invitrogen) and standard transfection protocols. This generated an estimated maximal transfection rate of 10 viral genomes per cell. GFP expression in control wells at 12 h post transfection was monitored by fluorescence microscopy, and the efficiency of plasmid/RNA uptake into cells was routinely judged to be approximately 80%.Infectious cDNA clones of full-length CVB3 strain 28 (CBV3), as well as of CVB3 containing a 5′ terminal deletion of 49 nt (TD-CVB3) , were us5 transfected cells to 105 untreated cells, down to one transfected/infected cell in a background of 105 untreated cells. Each cell dilution was centrifuged, the supernatant was removed, and pancreatic lysate was added to the cell pellets. Cells were incubated with pancreatic lysate on wet ice for 15 min, then flash frozen at −80 °C overnight before RNA extraction. RNA was isolated from cells in 50 µL of elution buffer using the MagMAX Viral RNA Isolation Kit (Invitrogen). cDNA was generated with the SuperScript III Reverse Transcriptase (Invitrogen) before analysis by qRT-PCR as described above.Alternatively, cells were infected with CVB3 virus at MOI (multiplicity of infection) = 10. Transfected cells and infected cells were incubated for 6 h at 37 °C in the presence or absence of 2 mM guanidine HCl before cells were harvested. To remove unabsorbed transfection RNA or virus, cells were washed five times with growth medium, then treated with RNase A (1 mg/mL) in medium for 30 min at 37 °C. Cells were again washed three times with PBS  and trypsinized. The transfected/infected cells were removed from the plate and mixed in serial one log dilution increments with untreated HeLa cells beginning at 10The ability of different RT-PCR methods to detect enterovirus RNA in pancreas was analyzed using pancreas homogenates spiked with different amounts of infectious CVB1 and CVA6 . The senTo evaluate the ability of detecting unencapsidated viral RNA, an experiment was carried out where transfected cells containing viral RNA were diluted into a background of non-transfected cells before recovery from pancreatic extracts A. Using In condition B, cells were infected 60 min to allow uptake, penetration, and release into the cytoplasm. Unadsorbed virus was washed away, and infected cells were diluted into a background of uninfected cells A. Then, RNA from the wild-type CVB3, as well as free RNA purified from terminally deleted CVB3, was tested for stability in pancreatic extract after incubation on ice for 15 min. Results indicate that almost no such terminally deleted RNA survived the interaction with pancreatic extract for a short period of time B.Since it is difficult to determine precisely how much transfected RNA is delivered into cells, we performed another experiment to compare viral recovery during replication versus non-replication conditions. Infections and transfections were carried out in HeLa cells as described above. Both wild-type CVB3 RNA and terminally deleted CVB3 RNA were transfected into the cells. To verify that viral RNA was introduced into the cells, we allowed six hours of incubation time for a measurable replication to occur B. Parall5 genome equivalents were introduced into the experiment. Notably, comparison of intact CVB3 RNA to terminally deleted CVB3 RNA indicated nearly a log lower detection level, likely due to the additional instability of terminally deleted CVB3 to cytoplasmic nucleases resulting from a loss of the stabilizing 5′ hairpin structure [Comparison of the detection of transfected wild-type CVB3 RNA versus terminally deleted CVB3 RNA from cultured cells indicates a further nine-fold loss of recovery under conditions where the replication was blocked A. There tructure .The present study demonstrates that detection of encapsidated enterovirus RNA by RT-PCR was not much affected by a 20-h-long incubation on ice in pancreatic homogenates prior to RNA extraction, while even a short incubation on ice led to dramatic decrease in the detection of unencapsidated viral RNA. In addition, the results showed that the sensitivity of different EV RT-PCR assays differed slightly depending on the assay itself and the sample type analyzed.All assays were capable of detecting very small levels of virus but did not always lead to amplicons when fewer than 1000 genomes per reaction were present, which is typical for this type of analysis. Importantly, the results indicate that pancreas tissue does not contain factors that significantly inhibit polymerase activity in molecular assays of this kind.An additional test in the experimental design was to see if the transport of pancreas on wet ice, which is an unavoidable part of the nPOD organ procurement procedure, adversely impacts the recovery of RNA from encapsidated virions. The data indicate that it does not, as the 20-h incubation with pancreatic extract on wet ice did not cause a significant decrease in the number of viral genomes detected. Thus, the transport of pancreas tissues on wet ice seems to be adequate for EV detection by RT-PCR, preventing the loss of viral RNA which easily happens at high temperatures . TogetheFurther experiments indicated that viral RNA with terminal deletions is rapidly destroyed by brief incubation on ice with pancreatic extract, and that detection of unencapsidated RNA was much lower than that of RNA encapsidated in complete virus particles. Under the conditions used, less viral RNA was recovered from transfected cells than from naturally infected cells. This could be partly due to the lack of nuclease-protective VPg  on transfected RNA and delivery via liposomes to cytoplasmic compartments that undergo lysosomal decay. These limitations make it difficult to quantitatively model the exact conditions of a low-level replicating terminally deleted CVB3 in cells. Nonetheless, the comparison of transfected RNA under non-replicating versus replicating conditions indicates that a proportion of transfected RNA is delivered to the correct cytoplasmic compartment to initiate genome replication. Under these conditions, we cannot precisely determine the limit of detection of unencapsidated CVB3 RNA in cells. However, taken together, the data indicate that detection would be approximately 100–1000-fold lower than that of encapsidated virion RNA. Detection of terminally deleted CVB3 RNA would add an additional eight-fold reduction of efficiency as compared to intact CVB3 RNA.Taken together, the results indicate that virus RNA that is protected inside capsids is stable to the RNase activities in pancreas extract, such that no substantial effect on virus RNA detection was noted. However, an increasing amount of data suggest that virus in the pancreas of T1D patients may be terminally deleted and replication-deficient. Such virus may have a much lower rate of encapsidation since enterovirus encapsidation efficiency is coupled to replication rates . FurtherIn conclusion, the study indicates that detection of unencapsidated viral RNA within rare cells in a population of uninfected cells, in an environment that contains pancreatic enzymes, is quite difficult. This does not derive from inhibitory effects of pancreatic factors on RT-PCR polymerases, but mostly from the exposure of viral RNA to RNase activities. The recovery of unencapsidated enterovirus RNA from such samples is not impossible but may require 100–1000 genome equivalents in each sample. The problem becomes more pronounced with terminally deleted EV that lacks a protective 5′ stem loop structure and undergoes encapsidation with greatly reduced efficiency . The res


Transfection, the process of introducing purified nucleic acids into cells, and viral transduction, viral-mediated nucleic acid transfer, are two commonly utilized techniques for gene delivery in the research setting. Transfection allows purified nucleic acid to be introduced into target cells through chemical-based techniques, nonchemical methods or particle-based methods, while viral transduction employs genomes or vectors based on adenoviruses, retroviruses (e.g. lentiviruses), adeno-associated viruses, or hybrid viruses. Transfected DNAs are often tested for potential effects on subsequent transduction, but it is not clear whether transfection itself rather than the particular nucleic acid being introduced might impact subsequent viral transfection. We observed a significant association between successfully transfected mobilized peripheral blood CD34+ human stem and progenitor cells (HSPCs) and permissiveness to subsequent lentiviral transduction, which was not evident in other cells such as 293 T cells and Jurkat cells. This association, apparently specific to CD34+ human stem and progenitor cells (HSPCs), is critical to both research and clinical applications as these cells are a frequent target of transfection and viral transduction owing to the durable nature of these cells in living systems. This finding may also present a significant opportunity to enhance the success of viral transduction for clinical applications. Transfection, the introduction of DNA into a cell by a variety of means, is widely used by researchers to deliberately genetically modify target cells. A large number of chemical and nonchemical techniques have been developed to allow for both transient and permanent transfection of target cells, including the use of calcium phosphate coprecipitates, basic polymers such as DEAE Dextran, lipophilic molecules such as lipofectamine, and electroporation –3. DespiCD34+ HSPCs are a cell type of particular interest for transfection and viral transduction as these cells are a durable and lasting source of circulating hematopoietic cells in vivo. A number of different approaches have been investigated to understand the basis of the observed limitations to transduction of these cells and improve efficiency. Investigators have exposed CD34+ HSPCs to various chemicals such as Cyclosporin A, Cyclosporin H, Rapamycin, stem cell factor, thrombopoietin, Flt3 ligand, interleukins, proteasome inhibitors and other agents before transduction with various levels of success .While the primary focus on transfection has been to study the impact on cells of the introduction of genetic material it would seem possible and even likely either that successfully transfected cells are different from cells that are resistant to successful transfection, or that transfected cells are modified in some way by the very process of being transfected –18. We w− vpr− nef−) with ZsGreen replacing luciferase, and the vesicular stomatitis virus glycoprotein (VSV-G) envelope (addgene cat#12259) [0 C for 2 h through a 25% sucrose cushion. Relative viral titer was determined through infection of permissive 293 T cell line with serial dilutions of the virus preparation, scoring for percent GFP positive by flow cytometry. For transfection we used pmCherry-C1 (Clontech cat# 632524) plasmid DNA in which a CMV promoter drives expression of the reporter. We selected to use a CMV promotor as this is a standard and effective promotor used in CD34+ cells for many applications including gene therapy targeting of human hematopoietic stem and progenitor cells (HSPCs) [We obtained 293 T cells and Jurkat cells from ATCC, and peripherally mobilized CD34+ HSPCs from individual donors from AllCells (pre-isolated). 293 T cells were cultured in 20 mm X 100 mm Falcon standard tissue culture dishes in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), penicillin, streptomycin, L-glutamine and 5% HEPES Buffer, and were passaged with trypsin using Gibco 0.25% Trypsin-EDTA (1X). Jurkat Cells were cultured in RPMI 1640 Medium containing 10% FBS. Human HSPCs were cultured in serum free media (X-VIVO 20), with stem cell factor (SCF), Flt3 ligand (FLT3L), and thrombopoietin (TPO) at 100 ng/ml. Lentiviral stocks for transduction were prepared by collecting culture supernatants from 293 T cells transfected with plasmid DNAs encoding a modified pNL4.3 HIV-1-based vector (AIDS Reagent Repository number 3418) (envt#12259) , 20. The (HSPCs) .https://bioscience.lonza.com). After transfection, 80 ul of culture media was added to the cuvette, and 50 ul of the cell suspension was removed and then placed in culture wells containing 200 ul of media. CD34+ cells were also transfected in cuvettes. 50,000 cells were suspended in 20 ul of transfection reagent, and 2 μg of pmCherry-C1 DNA were added. The cells were subjected to electroporation using the Lonza 4D-Nucleofector X Unit with the Lonza P3 Primary Cell 4D-Nucleofector X Kit S according to the Lonza Amaxa 4D-Nucleofector Protocol for unstimulated CD34+ cells (https://bioscience.lonza.com). After the transfection process, 180 ul of culture media was added to each cuvette well and mixed with the transfection solution. A total of 90 ul of media/solution was removed and placed in culture wells containing 160 ul of media.To test 293 T cells for effects of transfection, cells were plated in a 48-well plate at 50,000 cells per well. Twenty-four hours later the cells were transfected using lipofectamine. 100 ng of pmCherry-C1 plasmid DNA in 16 ul of Opti-MEM 1 Reduced Serum Medium (Gibco), was mixed with 2 ul of Lipofectamine 2000 at room temperature. Twenty minutes later the mixture was added to the cells in wells containing 250 ul of culture media to initiate the transfection . Jurkat In all cases, at twenty-four hours following transfection, cells were exposed to the lentiviral supernatants . The celReporter gene expression levels were quantified using flow cytometry on a BD Biosciences LSR-II at twenty-four hours post infection for 293 T and Jurkat cells, and at 72 h post infection for CD34+ HSPCs. We gated on viable cells (based ion FSC/SSC parameters) and then read out GFP and cherry red signals. Importantly, both reporter readouts demand that the introduced DNAs enter live cells and initiate reporter gene expression. Thus, all GFP and cherry red positive signals come from viable cells and will not score any nonproductive uptake of DNA or virus. We compared the efficiency of transduction (scored as percent GFP positive) of the cells that had not been successfully transfected (cherry red low) with those that were transfected (cherry red high) using flow cytometry. We found that transfection of 293 T cells with plasmid DNA using Lipofectamine 2000 followed by viral transduction at MOIs of 0.5 and 1 did not show significant impacts on susceptibility to viral transduction . Similar results were obtained over the multiple replicate experiments. Importantly, the selective difference in transduction efficiency is seen even though all the cells in the preparation were subjected to the identical treatment – the only distinction is the post-experiment filtering of the cells into the successfully transfected and the unsuccessfully transfected subsets.In contrast, we observed that after transfection of CD34+ cells with plasmid DNA by electroporation, the successfully transfected (cherry red high) cells showed an almost 3-fold increase in the percentage of successfully transduced (GFP+) cells at an MOI of 1 Fig. . ImportaThe results indicate a strong association between successful transfection of mobilized peripheral blood CD34+ human HSPCs and permissiveness to subsequent lentiviral transduction. It is notable that the enhanced susceptibility to transduction was only observed in HSPCs and not in transformed immortal fibroblastic or T cell lines. It is also important to note that the phenomenon cannot be attributed simply to the transfection process, because the increased susceptibility is only seen in the cell population that was successfully transfected and expressed the reporter gene, and not in the cells in the pool that were exposed to the same treatment but were not successfully transfected. The basis for the enhanced transduction is unknown, but there are several possible explanations. The cells most susceptible to transfection may represent a population subset with distinct abilities to traffic incoming nucleic acids or virion cores within the cytoplasm, into the nucleus, or to suitable locations within the nucleus for proviral integration. These steps are known to be inefficient in HSPCs . Close eOur prior work has demonstrated that the most significant block in human CD34+ cells is prior to nuclear entry and similar techniques involving quantitative PCR could be used to better understand the mechanism of this enhancement and the specific impact on various stages in the viral cycle . The obs