Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

Meganucleases can restore the reading frame of a mutated dystrophin.

Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog micro-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target micro-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the micro-dystrophin that restored dystrophin expression. MGNs also restored micro-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted micro-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

Characterization of rhesus monkey prostate specific antigen cDNA.

Using a RT-PCR approach, we obtained two overlapping cDNA clones containing the entire 1.5 kb sequence of rhesus monkey prostate specific antigen (rmPSA). The sequence obtained revealed an open reading frame of 261 amino acids. One potential N-glycosylation site was identified at Asn-78. The calculated molecular mass for the unglycosylated mature protein was 26,147 Da. Extensive amino acid homology (89%) was observed between rmPSA and its human counterpart. These results demonstrate that rhesus monkey and man prostate share a major biochemical component, and suggest that this animal species might be useful to answer specific questions related to human prostatic function and pathology.

Identification of glandular kallikrein in dog pancreas and determination of its tissue distribution.

In order to establish a formal link between previously purified canine urinary kallikrein and dog pancreatic kallikrein whose cDNA sequence has recently been published, we have isolated the pancreatic kallikrein from that animal species. Pancreatic cytosol proteins were sequentially subjected to chromatography on DEAE-Sepharose CL-6B and Concanavalin A-Sepharose, to an autolysis step and finally to two-dimensional gel electrophoresis. Kallikrein immunoreactive spots were identified with an antibody directed against canine urinary kallikrein. These proteins were isolated after electroblotting and the amino acid sequence of their NH2-terminal portion was determined by microsequencing. The sequence was found to be identical to the one deduced from pancreatic kallikrein cDNA. Using the same antibody and immunohistochemical procedures, kallikrein was found to be present in the pancreas, the salivary glands, the kidney, the colon, the lungs and the testis. These results thus confirm the molecular nature of a glandular kallikrein in the canine species.

Characterization of canine pancreas kallikrein cDNA.

Using a combination of primer extension and RT-PCR, the cDNA encoding a canine tissue kallikrein expressed in the pancreas was cloned and sequenced. The cloned 0.85 kbp cDNA contained a complete open reading frame encoding a polypeptide of 261 amino acids. The calculated molecular mass of the processed, unglycosylated, 237 amino acid protein was 26,428 Da. Its mRNA was expressed at high levels in the pancreas, kidney and submaxillary gland. The sequence of the encoded protein was highly homologous with canine prostatic arginine esterase (66%) and human renal/pancreatic kallikrein (74%). Therefore, the cloned cDNA encoded a previously uncharacterized canine kallikrein enzyme which was named dog renal/pancreatic kallikrein or dK2 according to the new nomenclature for kallikrein gene family members. Because of its specific pattern of tissue expression and the presence of all the amino acid residues necessary for kininogenase activity, we suggest that dK2 is the canine true tissue kallikrein.

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma.

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy.

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >>FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.

Prostaglandin biosynthesis, transport, and signaling in corpus luteum: a basis for autoregulation of luteal function.

The corpus luteum (CL) is a transient ovarian endocrine gland formed from the ovulated follicle. Progesterone is the primary secretory product of CL and is essential for establishment of pregnancy in mammals. In the cyclic female, the life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotrophic and luteolytic mediators. It is universally accepted that prostaglandin (PG) F(2a) is the luteolysin whereas PGE(2) is considered as a luteotropin in most mammals. New emerging concepts emphasize the autocrine and paracrine actions of luteal PGs in CL function. However, there is no report on selective biosynthesis and cellular transport of luteal PGE(2) and PGF(2alpha) in the CL of any species. We have studied the expression of enzymes involved in the metabolism of PGE(2) and PGF(2alpha), cyclooxygenase (COX)-1 and -2, PGE and F synthases, PG 15-dehydrogenase, and PG transporter as well as receptors (EP2, EP3, and FP) throughout the CL life span using a bovine model. COX-1, PGF synthase, and PG 15-dehydrogenase are expressed at constant levels whereas COX-2, PGE synthase, PG transporter, EP2, EP3, and FP are highly modulated during different phases of the CL life span. The PG components are preferentially expressed in large luteal cells. The results indicate that PGE(2) biosynthesis, transport, and signaling cascades are selectively activated during luteal maintenance. By contrast the PGF(2alpha) system is activated during luteal regression. Collectively, our results suggest an integrated role for luteal PGE(2) and PGF(2alpha) in autoregulation of CL function.

Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2.

Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and corpus luteum (CL). In ruminants, interferon-tau (IFNtau) of embryonic origin is recognized as the pregnancy recognition signal. Endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin, whereas PGE(2) is considered a luteoprotective or luteotrophic mediator at the time of establishment of pregnancy. The interplay between IFNtau and endometrial PGs production, transport, and signaling at the time of maternal recognition of pregnancy (MRP) is not well understood. We have studied the expression of enzymes involved in metabolism of PGE(2) and PGF(2alpha), cyclooxygenase-1 (COX-1) and COX-2, PG synthases (PGES and PGFS), PG 15-dehydrogenase, and PG transporter as well as PGE(2) (EP2 and EP3) and PGF(2alpha) receptors. IFNtau influences cell-specific expression of COX-2, PGFS, EP2, and EP3 in endometrium, myometrium, and CL in a spatio-temporal and tissue-specific manner, whereas it does not alter COX-1, PGES, PG 15-dehydrogenase, PG transporter, or PGF(2alpha) receptor expression in any of these tissues. In endometrium, IFNtau decreases PGFS in epithelial cells and increases EP2 in stroma. In myometrium, IFNtau decreases PGFS and increases EP2 in smooth muscle cells. In CL, IFNtau increases PGES and decreases EP3. Together, our results show that IFNtau directly or indirectly increases PGE(2) biosynthesis and EP2-associated signaling in endometrium, myometrium, and CL during MRP. Thus, PGE(2) may play pivotal roles in endometrial receptivity, myometrial quiescence, and luteal maintenance, indicating polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE(2) at the time of MRP. Therefore, the establishment of pregnancy may depend not only on inhibition of endometrial PGF(2alpha), but also on increased PGE(2) production in cattle.

Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy.

Prostaglandins (PGs) play important functions in the reproductive system, and PGE(2) appears necessary for recognition of pregnancy. We have found that PGE(2) is able to increase cAMP generation in the bovine endometrium. There are two PGE(2) receptors (EP), EP2 and EP4, that are coupled to adenylate cyclase to generate cAMP, but these receptors have not been studied in the bovine. We have cloned and characterized bovine EP2 and EP4 receptors and studied their expression in the uterus. The amino acid sequences of bovine EP2 and EP4 possess a high degree (>80%) of identity with the other mammalian homologs. EP2 is expressed in most tissues, and EP4 is expressed only in intestine and testis. EP2 mRNA and protein are expressed in endometrium and myometrium during the estrous cycle, whereas EP4 is undetectable. The Western analysis indicates that EP2 is maximally expressed in both endometrium and myometrium between d 10 and 18 of the estrous cycle. Immunohistochemical localization reveals that EP2 protein is expressed in all cell types of endometrium and myometrium. On d 18, pregnancy up-regulates EP2 protein, primarily in endometrial stroma and myometrial smooth muscle cells. In conclusion, EP2 is the major cAMP-generating PGE(2) receptor expressed and regulated in the bovine uterus during the estrous cycle and early pregnancy.

Progestin binding in testes from three siblings with the syndrome of male pseudohermaphroditism with testicular feminization.

We have found a specific binding protein for synthetic progestins 6,7-[3H]methyltrienolone (R1881) and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R5020) and in the testis cytosol from three "sisters" with the complete form of the testicular feminization syndrome. The binding component sediments in the 8S region of sucrose gradients. It is saturable. The apparent affinity constant (Ka) for R5020 was determined in two cases and found to be 1.8 and 0.6 X 10(8) M-1. The number of binding sites calculated from Scatchard plots is relatively high: 572 and 826 fmol/mg protein. Competition studies indicate that this putative receptor is specific for natural and synthetic progestins but not for 5 alpha-dihydrotestosterone and cortisol. Similar progestin binding could not be found in normal human and rat testes.

Demonstration of DNA binding factors interacting with a fragment of the canine prostate arginine esterase gene promoter.

We have studied, by the gel mobility shift assay, the interaction of DNA binding proteins with a fragment of the proximal promoter (from nucleotides -177 to -47) of the androgen-regulated canine prostate arginine esterase gene. Several shifted bands were obtained using nuclear extracts from various tissues. In the case of the prostate, the intensity of some of the shifted bands was decreased or increased when the extracts were prepared from animals that had been castrated 12 days earlier. Several of the DNA-protein complexes could be assigned to an interaction with part or all of the sequence GGGGGTGGGGG from-124 to -114. We also obtained evidence for the presence of protein(s) interacting with an Sp1 motif present in the same fragment. These results suggest that some ubiquitous factors different from the androgen receptors could be involved in the regulation of the arginine esterase gene.

High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate-specific antigen.

Using a synthetic oligonucleotide primer complementary to human prostate-specific antigen mRNA, we found that an additional sequence possibly similar to human glandular kallikrein-1 could be read by a primer-extension sequencing technique. We were able to confirm the identity of that additional sequence with another oligonucleotide primer complementary to a specific region of the human glandular kallikrein-1 mRNA sequence. Northern blot analysis with 2 oligonucleotide probes respectively specific for prostate-specific antigen and human glandular kallikrein-1 mRNAs showed that the length of both mRNAs was similar at 1.5 kb. The level of human glandular kallikrein-1 mRNA relative to that of prostate-specific antigen could be estimated as approx. 10-20%. This study constitutes the first evidence that the human glandular kallikrein-1 gene is expressed at a high level in a human tissue.

Nucleotide sequence of the androgen-dependent arginine esterase mRNA of canine prostate.

The nucleotide sequence of canine prostate arginine esterase mRNA was determined using a 400 bp cDNA clone and primer-extended cDNA transcripts for the 5'-coding and noncoding regions. The mRNA contains 864 nucleotides encoding a protein of 236 amino acids preceded by 24 amino acids which constitutes both the signal and the zymogen peptides. The sequence indicates the presence of one potential glycosylation site. A high degree of homology was found between the canine enzyme and other members of the kallikrein family including human prostate specific antigen. The protein appears to be specified by a single gene.

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing.

Canine prostate fluids and seminal plasma contain a major androgen-dependent protein which was identified as a proteolytic enzyme exhibiting an Arg-esterase activity. This protease, as characterized, is shown to be present as a two-chain structure held together by at least one disulfide bridge and composed of approximately 220 amino acids. Amino acid sequence determination of both chains has revealed a clear homology to other known amino acid sequences of serine proteases. Furthermore, the comparison of the presented 58 amino acids of the Arg-esterase with the other sequences revealed a very strong homology (larger than 50%) to members of the kallikrein family. The two chain structure could thus result from autolysis of a single chain enzyme in the 'kallikrein autolysis loop'. Amino acid composition of the canine prostatic enzyme suggests that it is related, but not identical, to pancreatic canine kallikrein.

Transcriptional regulation of dog prostate arginine esterase gene by androgens.

These studies were designed to define the molecular events involved in the modulation of dog prostate arginine esterase gene expression following short castration intervals and androgen treatment. Arginine esterase enzymatic activity and protein levels decreased about 50% 24 h after castration. Thereafter, a more progressive decrease was observed, resulting in 2-4-fold lower levels in 12-day castrates than in the intact controls. Total prostatic arginine esterase mRNA levels slowly decreased during the first five days after castration but more abruptly thereafter and were about 150-fold lower in 12-day castrated animals. By contrast, in isolated prostatic nuclei, levels of arginine esterase RNA precursors and mature transcripts rapidly fell following orchiectomy, with a 50-70% decrease 24 h after castration. Nuclear run-on experiments confirmed that the latter effects were the result of decreased arginine esterase gene transcription. All these changes could be at least partially reversed by administration of testosterone cypionate. Furthermore, no striking modifications in the proportion of epithelial/stromal cells in the prostatic tissue were observed following orchiectomy. These results show that castration and androgens exert very rapid effects on the gene expression of arginine esterase, and that the regulation occurs at the transcriptional level.

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA.

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.

Characterization and expression of the prostatic arginine esterase gene, a canine glandular kallikrein.

The prostatic arginine esterase gene was isolated from a genomic library prepared with dog liver DNA in lambda EMBL3. The selected clone contained an insert of approximately 17 kb which included the whole coding portion of arginine esterase mRNA (5 exons plus 4 introns), 2 kb upstream from the initiation site and 12 kb downstream from the polyadenylation site. The intron-exon boundaries were identical to all known mammalian kallikrein genes. The deduced amino acid sequence indicated a high degree of identity (51-61%) with other kallikreins expressed not only in the prostate but also in the pancreas of various animal species. The 5'-flanking sequences contained potential regulatory elements such as a variant TATA box (TTTAAA), a CCAAT box, a SP1 transcriptional factor binding site (GGGCGG), and two TGTCCT motifs resembling glucocorticoid response elements. Southern blot analysis with an amplified cDNA fragment of 487 bp corresponding to the 5' portion of the mRNA and with a DNA probe from a different portion of the arginine esterase gene indicated the presence of two to three homologous genes in the canine genome while in a previous study a single band was detected using a 400-bp arginine esterase cDNA corresponding to the 3' portion of the mRNA. These results suggest that the arginine esterase gene belongs to a small kallikrein gene family. Arginine esterase mRNA is expressed primarily in the prostate but also at an extremely low level (approximately a thousandfold less) in several other tissues including the liver, the gracilis thigh muscle, the kidney, and the pancreas.

Actin and creatine kinase mRNAs in rat levator ani and vastus muscles as a function of androgen status.

The plasticity of two selected mRNAs was studied in two typical fast-twitch muscles at different time intervals after orchiectomy (GDX). The levator ani muscle of the rat (LA) is exquisitely sensitive to androgens, whereas the superficial vastus lateralis (SVL) lacks such sensitivity. In vitro translation of RNA isolated from both tissues indicated that actin was among the most repressed proteins of the LA at day 10 postsurgery (GDX-10 days), whereas the template activity of the SVL mRNAs remains virtually unmodified. We used an available actin cDNA and demonstrated that the expression of the LA actin message is reduced by 85% in GDX-10 days and can be recovered after testosterone propionate (TP) injections (GDX + TP). In contrast, the actin expression in SVL remains constant up to day 20 postsurgery. In the LA, the expression of creatine kinase (CK) mRNA was increased 140% in GDX-5 days and decreased 34 and 17% in GDX-10 days and GDX-20 days, respectively, although the measured CK activity, as well as the in vitro translation of the message, remained elevated in those two latter groups. Control level of the CK mRNA expression was recovered in the GDX + TP group. Again, the expression of the message was unchanged in SVL, suggesting that the protein synthesis of this skeletal muscle is far less sensitive to androgen deprivation than that of the LA muscle.

Neutral alpha-1,4-glucosidase in human seminal plasma: molecular forms in varicocele and after vasectomy.

Neutral alpha-1,4-glucosidase catalyzes the breakdown of oligosaccharides in several tissues including the reproductive organs, and we have demonstrated the presence of two molecular forms (F1 and F2) of the enzyme in human seminal plasma. The identification of these forms can be achieved by sucrose density gradient analysis and/or electrophoresis in the presence of detergents. Individuals with normal sperm analyses or affected by a varicocele, thus showing normo- or oligoasthenozoospermia, show a similar prevalence of F1 only and F1 + F2 forms, while the presence of F2 alone becomes obvious following vasectomy. In conclusion, molecular forms of the enzyme do not appear to be indicative of the presence of a varicocele, but they may reflect modifications in the secretory function of specific reproductive organs (prostate) or glands (seminal vesicles), as observed in the course of an obstructive abnormality at the epididymal or vas deferens level.