Rina Zilkha-Falb

The role of laquinimod in modulation of the immune response in relapsing–remitting multiple sclerosis: Lessons from gene expression signatures

Laquinimod, is a potential oral immunomodulatory drug, for relapsing-remitting multiple sclerosis (RRMS). We analyzed the blood-transcriptional changes in RRMS patients (who participated in the ALLEGRO clinical trial) at one and six months after laquinimod treatment using gene expression microarrays. The molecular effects of laquinimod were enhanced by duration of treatment and showed down-regulation of inflammatory responses mainly via TGFb signaling, and of pro-inflammatory cytokines as well as of cellular movement, including adhesion, migration and leukocyte extravasation signaling. Our results demonstrate that laquinimod suppresses inflammation through down-regulation of inflammatory cytokines and arrest of leukocyte extravasation and thereby could attenuate disease activity in RRMS patients.

Polymerase I pathway inhibitor ameliorates experimental autoimmune encephalomyelitis.

Applying high throughput gene expression microarrays we identified that the suppression of polymerase 1 (POL1) pathway is associated with benign course of multiple sclerosis (MS). This finding supports the rationale for direct targeting of the POL1 transcription machinery as an innovative strategy to suppress MS. To evaluate the effects of a specific polymerase I inhibitor (POL1-I) on experimental autoimmune encephalomyelitis (EAE), we immunized female C57BL/6J mice (8 weeks) with MOG35-55/CFA. A new POL1-I was administered at a daily dose of 12.5mg/kg body weight by oral gavage either from the day of immunization until disease onset (EAE score 1.0, immunization model), at disease onset (EAE score=1.0) for the following 14 days (treatment model), or by alternate daily dose of 25.0mg/kg body weight, by oral gavage from the day of immunization for the following 25 days (combined model). POL1-I remarkably suppressed EAE in the immunization model; while in the Vehicle group the onset of EAE occurred on day 10.0±0.4 with maximal clinical score of 3.2±0.2, in the POL1-I treated mice onset was significantly delayed and occurred on day 16.9±1.1 (p=0.001), and maximal disease score 2.0±0.1 was reduced (p=0.004). In the treatment model POL1-I treatment significantly reduced disease activity; maximal score was 2.0±0.5 while in the Vehicle group it reached a mean maximal score of 3.9±0.1, (p=0.0008). In the combined model, POL1-I treatment completely inhibited disease activity. The effect of POL1-I treatment was modulated through decreased expression of POL1 pathway key-related genes LRPPRC, pre-RNA, POLR1D and RRN3 together with activation of P53 dependent apoptosis of CD4+ splenocytes. Our findings demonstrate that POL1 pathway inhibition delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

RAM-589.555 a new Polymerase-1 inhibitor as innovative targeted-treatment for multiple sclerosis

Targeting Polymerase-1 (POL1) transcription machinery is a new strategy for suppression of multiple sclerosis (MS) disease activity that is based on suppression of ribosomal biogenesis and subsequent activation of apoptosis. We developed an oral POL1 inhibiting compound RAM-589.555, that suppress ribosomal biogenesis as an innovative therapeutic approach to ameliorate MS. RAM-589.555 shows high permeability, specificity to POL1 pathway, ability to induce apoptosis and to inhibit proliferation and viability of activated lymphocytes both in-vitro and in-vivo. Moreover, oral administration of RAM-589.555 blocks ribosomal RNA transcription and significantly suppresses and ameliorates experimental autoimmune encephalomyelitis (EAE).

Experimental Autoimmune Encephalomyelitis Ameliorated by Passive Transfer of Polymerase 1-Silenced MOG35-55 Lymphatic Node Cells: Verification of a Novel Therapeutic Approach in Multiple Sclerosis

In the current study, we present an innovative concept based on the knowledge that enhancing naturally occurring biological mechanisms is effective in preventing neuronal damage and maintaining low disease activity in about 15% of multiple sclerosis (MS) patients presenting the benign type of MS. Recently, we have demonstrated that low disease activity in benign MS is associated with suppression of RNA polymerase 1 (POL1) pathway; therefore, targeting POL1 transcription machinery as a strategy for suppressing active forms of MS is suggested. To further establish our approach, we aimed to suppress POL1 pathway by silencing of the POL1-related RRN3, POLR1D and LRPPRC genes in specific MOG35-55-activated lymphocytes and assess their capacity to induce experimental autoimmune encephalomyelitis (EAE) by passive transfer. We have demonstrated that silencing of specific POL1 pathway-related genes significantly decreased viability and increased the proportion of CD4+/AnnexinV+/PI+ apoptotic cells in MOG35-55-primed lymphocytes. POL1-gene silencing significantly decreased the proportion of CD4+IL17+ and increased proportion of CD4+IL10+ and CD4+TNFa+ lymphocytes that occurred simultaneously with over-presentation of Treg CD4+CD25+FoxP3+ cells. Passive transfer of MOG35-55-primed lymphocytes after POL1-gene silencing suppressed EAE development in mice as demonstrated by delayed onset and peak of disease accompanied by significantly lower maximal and cumulative EAE scores. Our study supports a basis for direct targeting of POL1 transcription pathway as a strategy for selective induction of apoptosis and suppression of inflammation in EAE and consequently paves the way for innovative and targeted MS therapeutic strategy that is based on naturally existing biological mechanism.

Laquinimod suppresses Genes encoding Cell migration in Multiple Sclerosis

Multiple sclerosis (MS) is an immune mediated disorder of the central nervous system. T-cells and other immune effector cells play crucial roles in MS pathogenesis by crossing the brain–blood barrier and consequently destroying the myelin sheat and axons. Fingolimod (FTY720) is an oral sphingosine-1-phosphate (S1P) receptor modulator, approved for treatment of MS. Resting T and B lymphocytes express elevated levels of S1P receptor and lymphocyte migration from the lymphoid organs to the brain depends on the activity of this receptor. Recent evidence suggests that fingolimod does not only prevent lymphocyte migration to the central nervous system but also exhibit other mechanisms of action. To investigate the potential effector functions of fingolimod on cytokine, chemokine and soluble adhesion molecule production, levels of IL-17A, IL-10, IL-6, IL-13, IFN-γ, TNFα, IL-4, IL-12p40, soluble VLA4 (sVLA4), sVCAM1, sICAM, CXCL13, CCL2 and IL-8 were measured by ELISA in sera obtained before (S0) and three months after the onset (S3m) of fingolimod treatment in 10 MS patients. Serum sVLA4 levels were significantly decreased in all MS patient’s sera after treatment and sVLA4 levels were significantly lower in S3m sera as compared to S0 sera. By contrast, levels of other examined factors did not show any statistical difference among S0 and S3m sera. These results suggest that in contrast with other immunomodulating agents, shortterm fingolimod treatment does not affect Th1 and Th17 immunities. Reduction of sVLA4 by fingolimod treatment may contribute to prevention of lymphocyte crossing through blood–brain barrier and thus amelioration of MS symptoms.