Paola Rimessi

Preclinical PK and PD Studies on 2 '-O-Methyl-phosphorothioate RNA Antisense Oligonucleotides in the mdx Mouse Model

Antisense oligonucleotides (AONs) are being developed as RNA therapeutic molecules for Duchenne muscular dystrophy. For oligonucleotides with the 2′-O-methyl-phosphorothioate (2OMePS) RNA chemistry, proof of concept has been obtained in patient-specific muscle cell cultures, the mouse and dog disease models, and recently by local administration in Duchenne patients. To further explore the pharmacokinetic (PK)/pharmacodynamic (PD) properties of this chemical class of oligonucleotides, we performed a series of preclinical studies in mice. The results demonstrate that the levels of oligonucleotides in dystrophin-deficient muscle fibers are much higher than in healthy fibers, leading to higher exon-skipping levels. Oligonucleotide levels and half-life differed for specific muscle groups, with heart muscle showing the lowest levels but longest half-life (~46 days). Intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) delivery methods were directly compared. For each method, exon-skipping and novel dystrophin expression were observed in all muscles, including arrector pili smooth muscle in skin biopsies. After i.v. administration, the oligonucleotide peak levels in plasma, liver, and kidney were higher than after s.c. or i.p. injections. However, as the bioavailability was similar, and the levels of oligonucleotide, exon-skipping, and dystrophin steadily accumulated overtime after s.c. administration, we selected this patient-convenient delivery method for future clinical study protocols.

HIV-1 Tat Protein Modulates the Generation of Cytotoxic T Cell Epitopes by Modifying Proteasome Composition and Enzymatic Activity

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposis sarcoma (KS), primary effusion lymphoma and Castelmans disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.

A human melanoma metastasis-suppressor locus maps to 6q16.3-q23

Loss, deletion or rearrangement along large portions of the long arm (q‐arm) of chromosome 6 occurs in >80% of late‐stage human melanomas, suggesting that genes controlling malignant characteristics are encoded there. Metastasis, but not tumorigenicity, was completely suppressed in the human melanoma cell line C8161 into which an additional intact chromosome 6 had been introduced by microcell‐mediated chromosome transfer. Our objective was to refine the location of a putative metastasis suppressor gene. To do this, we transferred an intact (neo6) and a deletion variant [neo6qdel; neo6(del)(q16.3‐q23)] of neomycin‐tagged human chromosome 6 into metastatic C8161 subclone 9 (C8161.9) by MMCT. Single cell hybrid clones were selected in G‐418 and isolated. Following verification that the hybrids retained the expected regions of chromosome 6 using a panel of polymorphic sequence‐tagged sites, the hybrids were tested for tumorigenicity and metastasis in athymic mice. As reported previously, intact, normal chromosome 6 suppressed metastasis whether tumor cells were injected i.v. or into an orthotopic (i.e., intradermal) site. In contrast, metastasis was not suppressed in the neo6qdel hybrids. Tumorigenicity was unaffected in hybrids prepared with either chromosome 6 donor. These data strongly suggest that a human melanoma metastasis suppressor locus maps between 6q16.3‐q23 (≈40 cM). Int. J. Cancer 86:524–528, 2000.

Phenotypic and genotypic heterogeneity in transthyretin-related cardiac amyloidosis: Towards tailoring of therapeutic strategies?

Transthyretin-related hereditary amyloidosis (ATTR) is genotypically/phenotypically heterogeneous. We investigated myocardial involvement in ATTR in a cohort of patients with a wide range of mutations. Clinical/echocardiographic follow-up of 41 consecutive symptomatic ATTR patients from a single referral center was analyzed according to TTR mutation. Diagnosis was based on histology, immunohistochemistry and genotyping. Median follow up was 40 months (range 8–120). Among the 12 different mutations identified, Val30Met was found in 10 patients and Glu89Gln in seven. Compared with Val30Met, Glu89Gln was associated with higher LV mass index, lower left ventricular ejection fraction and shorter E-wave deceleration time. All Glu89Gln carriers had cardiomyopathy, which was more severe (for left ventricular thickness, left ventricular mass and restrictive pathophysiology) than in the six affected Val30Met patients. Glu89Gln was independently associated with higher risk of major cardiovascular events among cardiomyopathy patients. This follow-up study of ATTR patients carrying a wide range of mutations indicates that (1) cardiac involvement is a very important component of phenotypic expression; and (2) genotype is an important source of heterogeneity in myocardial involvement, with Glu89Gln being associated with a severe, heart-driven prognosis. We think that combined heart–liver transplantation could be considered for Glu89Gln carriers with established, morphologically severe cardiomyopathy.

Association of CYP1B1 with hypersensitivity induced by Taxane therapy in breast cancer patients

Taxanes represent a group of anticancer drugs with a wide range of activity against breast cancer. Therapy side effects include haematologic toxicity (neutropenia, leucopenia), peripheral neuropathy and hypersensitivity, and demonstrate inter-individual variations. Since it is known that three genes are implicated in Taxane turnover, namely ABCB1 in the transport, CYP2C8 in the metabolism and CYP1B1 in the activity, we explored the association among polymorphisms (single nucleotide polymorphisms, SNPs) in these three genes and the occurrence of Taxane-induced toxicity. We studied 95 patients affected by breast cancer and under treatment with Taxanes as adjuvant, metastatic or neo-adjuvant therapy. We genotyped them for SNPs in the CYP2C8 (alleles *1, *2, *3 and *4), CYP1B1 (alleles *1 and *3) and ABCB1 (1236 C>T; 2677 G>T/A; 3435 C>T) genes by real-time PCR assay. We observed a significant association between the CYP1B1*3 allele and a lower occurrence of hypersensitivity reactions to Taxane treatment. We speculate that the highest production of 4-hydroxyestradiol (4-OHE2) metabolite by CYP1B1*3 allele could increase the formation of the 4-OHE2-Taxane adduct and possibly inhibit Taxane toxicity. We suggest that CYP1B1 might affect Taxane hypersensitivity therefore representing, if confirmed in a large cohort of patients, an exploratory hypersensitivity predictive biomarker.

Growth arrest and suppression of tumorigenicity of bladder carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9

Wild‐type P16/CDKN2 (p16INK4A, MTSI) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder‐carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild‐type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over‐expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2‐gene expression is modulated by the physiological control of chromosomal regulatory sequences. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder‐carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32–33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor‐suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder‐carcinoma cells when it is over‐expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder‐cancer progression.

Transcription Pattern of Human Herpesvirus 8 Open Reading Frame K3 in Primary Effusion Lymphoma and Kaposi's Sarcoma

ABSTRACT Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castlemans disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposis sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals.

Multiple exon skipping and RNA circularisation contribute to the severe phenotypic expression of exon 5 dystrophin deletion

Deletion and duplication of one or more exons in the dystrophin gene account for 70% of patients with Duchenne and Becker muscular dystrophies (DMD and BMD) and other allelic clinical entities such as raised serum creatine kinase and X linked dilated cardiomyopathy (XLDC).1 The severity of the resulting phenotype can be generally predicted by whether these mutations lead to translation frame disruption and premature termination of protein synthesis.2 Nevertheless, the occurrence of severely affected patients with in frame deletions as well as mild phenotypes associated with frameshift, indicate that factors other than the frame disruption should contribute to the clinical severity. Exceptions to the “frame rule” are found in about 8% of patients with mutations occurring both at the 5′ and 3′ end of the dystrophin gene,1,3,4 although they seem to predominate in the 5′ region.4 Despite extensive clinical, immunocytochemical, and transcriptional studies, the basis of genotype-phenotype correlation in these “atypical” cases remains controversial and its clarification will surely provide relevant information about normal and abnormal dystrophin function. Several reports suggest a role for alternative splicing in altering the clinical phenotype by modulating the editing of the translation reading frame.3,5,6 Patients with BMD carrying the frameshift deletions of exons 3–7 and 45 show alternative splicing phenomena theoretically restoring the reading frame.5,7 However, the relevance of these events in contributing to a milder phenotype is still unclear.3,8,9 Supporting the bridging role of the dystrophin splicing machinery as active modulator between genotype (deletion mutation) and protein production, cell specific somatic exon skipping has been documented in skeletal muscle revertant fibres in DMD.10 ### Key points

Dystrophin restoration in skeletal, heart and skin arrector pili smooth muscle of mdx mice by ZM2 NP-AON complexes

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2–AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2–AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as α-, β-, γ- and δ-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.