Genetic Correction of Stem Cells in the Treatment of Inherited Diseases and Focus on Xeroderma Pigmentosum

International audienceSomatic stem cells ensure tissue renewal along life and healing of injuries. Their safe isolation, genetic manipulation ex vivo and reinfusion in patients suffering from life threatening immune deficiencies (for example, severe combined immunodeficiency (SCID)) have demonstrated the efficacy of ex vivo gene therapy. Similarly, adult epidermal stem cells have the capacity to renew epidermis, the fully differentiated, protective envelope of our body. Stable skin replacement of severely burned patients have proven life saving. Xeroderma pigmentosum (XP) is a devastating disease due to severe defects in the repair of mutagenic DNA lesions introduced upon exposure to solar radiations. Most patients die from the consequences of budding hundreds of skin cancers in the absence of photoprotection. We have developed a safe procedure of genetic correction of epidermal stem cells isolated from XP patients. Preclinical and safety assessments indicate successful correction of XP epidermal stem cells in the long term and their capacity to regenerate a normal skin with full capacities of DNA repair

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na(+) and K(+) channels. APETx2 reversibly inhibits rat ASIC3 (IC(50)=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC(50)=117 nM), while it has less affinity for ASIC1b+3 (IC(50)=0.9 μM), ASIC1a+3 (IC(50)=2 μM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC(50) of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels

Black mamba venom peptides target acid-sensing ion channels to abolish pain

International audiencePolypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia

Basal Cell Carcinoma in Gorlin’s Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

International audienceBasal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients

NBCCS fibroblasts alter morphology and differentiation of WT epidermis.

<p>WT keratinocytes were seeded onto dermis equivalents comprising skin fibroblasts isolated from either a WT (WT1) donor or from two independent NBCCS patients (NBCCS6 and NBCCS10). H&E staining reveals the absence of cornified layers (CL) and slight epidermal thinning in NBCCS OSC. Note increased accumulation of Laminin B1 and β1 Integrin in the basal layer (BL) and the para basal layers of epidermis. Star (*) points dermo epidermal cleft between the dermal and the epidermal compartments. Also note delayed expression of Keratin 10 (K10) and absence of expression of Loricrin (Lor) in NBCCS OSC; PI, nuclei counter staining using propidium iodide. Dash lines delineate the dermo epidermal junction. Bar: 110 μm</p

Neutralizing SHH in organotypic skin cultures attenuates alterations of WT epidermis in the presence of NBCCS fibroblasts.

<p>OSC comprising either WT (WT1) or NBCCS fibroblasts (NBCCS6 or NBCCS10) were developed in the presence of either the 5E1 anti-SHH blocking mAb (5E1 mAb) or an isotype-matched (anti-Myc 9E10) control antibody (Control mAb). OSC sections were stained with either H&E, or immunolabeled with the anti-β1 Integrin mAb. Note that developing OSC in the presence of the 5E1 antibody improved epidermal atrophy (as pointed out by black arrows) and β1 Integrin delocalization otherwise observed in the presence of NBCCS fibroblasts. Bar: 110 μm</p

Culture supernatants from NBCCS fibroblasts promote SA-β-Gal activity in WT but not in transformed (E6-E7) or tumoral (SCC13) keratinocytes.

<p>(A) SA-β-Gal activity in WT1, WT1 E6-E7, and SCC13 keratinocytes after treatment with either non conditioned medium (NCM), culture supernatant conditioned by WT (WT1), or NBCCS (NBCCS6 and NBCCS10) fibroblasts. (B) SA-β-Gal positive cells were blind counted by three independent investigators. Bars represent the confidence intervals (α = 0.05). Number of SA-β-Gal positive cells was increased in WT keratinocytes treated with NBCCS supernatants. No increase of SA-β-Gal positive cells was detected in both WT1 E6-E7 and SCC13 keratinocytes.</p

Abrogation of P53 in NBCCS keratinocytes confers invasive properties in the presence of NBCCS fibroblasts.

<p>(A) Western blot analysis of P53 showed a strong attenuation in WT (WT1), NBCCS6 and NBCCS10 keratinocytes transduced with a retrovirus expressing the HPV16 E6 and E7 oncoproteins. (B) Representative images of H&E coloration of paraffin-embedded sections of organotypic invasion assays composed of either WT E6-E7 or NBCCS E6-E7 keratinocytes overlaying a dermis equivalent comprising either WT or NBCCS fibroblasts. Bar: 200 μm. (C) Invasion index of keratinocytes from assays as in (B). Values represent the average of 3 total sections from two independent organotypic cultures. Bars represent the confidence intervals, α = 0.05.</p

NBCCS fibroblasts stimulate <i>PATCHED1</i> transcription in WT keratinocytes.

<p>(A), <i>PATCHED1</i> and <i>GAPDH</i> mRNAs amounts expressed by WT keratinocytes cultured with either WT or NBCCS fibroblasts (NBCCS6 or NBCCS10) were determined after semi-quantitative PCR and agarose gel electrophoresis (upper panel). Relative <i>PATCHED1</i> mRNA levels were increased in WT keratinocytes (WT1) treated with culture media derived from NBCCS fibroblasts (NBCCS6 and NBCCS10) (lower panel). (B), Activity of the <i>PATCHED1</i> promoter in WT keratinocytes cultured in media conditioned from either WT or NBCCS fibroblasts (NBCCS6 or NBCCS10). Note the substantial increase of the relative transcriptional activity of the <i>PATCHED1</i> promoter in WT keratinocytes treated with culture media conditioned by NBCCS fibroblasts. Experiments were performed two times in triplicates, p<0.09.</p

SHH secretion depends on the presence of both fibroblasts and keratinocytes and is further increased in the presence of NBCCS fibroblasts.

<p>(A), Western blot analysis of N-SHH secreted from either WT (WT1) or NBCCS (NBCCS6 or NBCCS10) fibroblasts. Secretion of N-SHH was not detectable in culture supernatants of WT and NBCCS fibroblasts. (B), Western blot analysis of N-SHH secreted from co-cultures of WT keratinocytes with WT (WT1) or NBCCS fibroblasts (NBCCS6 or NBCCS10). N-SHH became slightly detectable in the presence of WT fibroblasts (WT1). N-SHH amount was substantially increased in the presence of NBCCS fibroblasts (NBCCS6, NBCC10). Lower panel: quantitation of N-SHH in culture supernatant obtained under the circumstances described for the upper panel. Note that detection of SHH is limited to the N-SHH processed protein (22 kDa, arrow).</p