Intratumoral Delivery of Plasmid Interleukin-12 Via Electroporation Leads to Regression of Injected and Non-Injected Tumors in Merkel Cell Carcinoma

Purpose: Interleukin-12 (IL12) promotes adaptive type I immunity and has demonstrated antitumor efficacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer. Experimental Design: Fifteen patients with MCC with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N = 3) received one cycle before definitive surgery in week 4. Patients with metastatic MCC (cohort B, N = 12) received up to four cycles total, administered at least 6 weeks apart. Serial tumor and blood samples were collected. Results: All patients successfully completed at least one cycle with transient, mild (grades 1 and 2) AEs and without significant systemic toxicity. Sustained (day 22) intratumoral expression of IL12 protein was observed along with local inflammation and increased tumor-specific CD8+ T-cell infiltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in cohort B, with 2 patients experiencing durable clinical benefit (16 and 55+ months, respectively). Two cohort A patients (1 with pathologic complete remission) were recurrence-free at 44+ and 75+ months. Conclusions: I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL12 protein and local inflammation led to systemic immune responses and clinically meaningful benefit in some patients. Gene electrotransfer, specifically i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer

In vivo functional analysis of the human NF2 tumor suppressor gene in Drosophila.

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes

Molecular biology of male gametogenesis

International audiencePollen development has been studied at a molecular level in several systems that are amenable to genetic or transgenic analysis. We have characterized several tomato genes that are expressed late in pollen development. Our goals in this research were 1) to determine the cis- and tran-acting factors that mediate pollen expression, and 2) to determine the functions of the proteins encoded by these genes. We currently favor the hypothesis that pollen-specific gene expression is mediated in a combinatorial manner. Antisense experiments have indicated an important role for the LAT52 protein during pollen hydration

In Vivo Functional Analysis of the Human <i>NF2</i> Tumor Suppressor Gene in Drosophila

The proper control of tissue growth is essential during normal development and an important problem in human disease. Merlin, the product of the Neurofibromatosis 2 tumor suppressor gene, has been extensively studied to understand its functions in growth control. Here we describe experiments in which we used Drosophila as an in vivo system to test the functions of the normal human NF2 gene products and patient-derived mutant alleles. Although the predominant NF2 gene isoform, isoform 1, could functionally replace the Drosophila Merlin gene, a second isoform with a distinct C-terminal tail could not. Immunofluorescence studies show that the two isoforms have distinct subcellular localizations when expressed in the polarized imaginal epithelium, and function in genetic rescue assays correlates with apical localization of the NF2 protein. Interestingly, we found that a patient-derived missense allele, NF2L64P, appears to be temperature sensitive. These studies highlight the utility of Drosophila for in vivo functional analysis of highly conserved human disease genes.</p

Molecular biology of male gametogenesis

International audiencePollen development has been studied at a molecular level in several systems that are amenable to genetic or transgenic analysis. We have characterized several tomato genes that are expressed late in pollen development. Our goals in this research were 1) to determine the cis- and tran-acting factors that mediate pollen expression, and 2) to determine the functions of the proteins encoded by these genes. We currently favor the hypothesis that pollen-specific gene expression is mediated in a combinatorial manner. Antisense experiments have indicated an important role for the LAT52 protein during pollen hydration

qPCR analysis of effect of hNF2 expression on endogenous Merlin expression.

1<p>Fold expression change compared to control genotype, <i>ApGal4/+.</i></p

Genetic rescue by hNF2 alleles.

1<p>Average of three or more independent lines.</p>2<p>Using two of the insertions tested in the one-dose experiments.</p

Mutations in <i>hNF2</i> alter the subcellular localization of the Merlin protein expressed in Drosophila.

<p>Confocal optical sections of wing imaginal epithelia, either cutting deeply into the epithelium (‘Cross section’) or tangentially through the apical surface of the epithelium, are presented for each allele or isoform. A–B) NF2 isoform 1 is primarily localized to the apical membrane (arrows) of these polarized epithelial cells, and adopts a punctate appearance at the apical cortex that is not clearly associated with cell boundaries (see inset at right). In addition, some punctate staining is observed basally (arrowheads in A). C–D) In contrast isoform 2, while still primarily apical (arrows, C), appears associated with cell boundaries (outlining the periphery of the apical ends of cells in D) and does not form punctae. The localizations of the K413E (E–F) and L64P (G–H) point mutations appear similar to that of isoform 1. The Δ2-3 deletion allele (I-J) appears localized to the lateral cell cortex, somewhat similar to isoform 2, but less strongly apically localized (arrows mark apical; also note staining throughout the basolateral domain in I). NF2^1-356 (K–L) is substantially cytoplasmic and fails to accumulate apically while NF2^351-595 (M–N) shows some apical enhancement (arrows in M). Both truncations display substantial cytoplasmic localizations, as indicated by the tangential views (compare L and N to J).</p

Ectopic expression of hNF2 isoforms 1 and 2 has different effects on Drosophila wing development.

<p>Two transgenic copies of each isoform were expressed in the dorsal half of the developing wing blade under the control of the <i>apterous</i>-Gal4 driver. The apterous-Gal4 driver itself has little or no effect on wing development (A–B), while expression of isoform 1causes some ectopic vein formation, particularly along the second and fifth wing veins (C–D). Expression of isoform 2 (E–F) causes more severe ectopic vein formation that includes veins 2, 3 and 5. In addition, expression of this isoform causes a downward cupping of the wing blade (data not shown).</p