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Literature Supporting The Use Of Dietary Nucleotides
 

Literature Supporting The Use Of Dietary Nucleotides

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    Literature Supporting The Use Of Dietary Nucleotides Literature Supporting The Use Of Dietary Nucleotides Presentation Transcript

    • Literature Supporting The Use Of Dietary Nucleotides Dr. Garry Gordon MD, DO, MD(H)
      • “ Dietary nucleotides have been shown to have important effects on several components of the immune system: they may enhance intestinal absorption of iron; they affect lipoprotein and long-chain polyunsaturated fatty acid metabolism; they may alter intestinal flora; and they have been demonstrated to have trophic effects on the intestinal mucosa and liver in several experimental situations. Clinical studies have shown nucleotide supplementation of infant formula reduces the incidence of diarrheal episodes…”
      • Cosgrove M. Nucleotides. Nutrition 1998;14:748–51.
      DIETARY NUCLEOTIDES
    • RNA CONTAINING FOODS CURRENTLY ON THE MARKET
      • IMPACT ( novartis/sandoz )
      • Primary indications:
              • Trauma
              • Major Surgery
              • Infections of pneumonia
              • Cancer
              • Ventilator dependent
              • Burn Injury
      • “ There have been numerous studies of the clinical, immunological, nutritional and biochemical effects of IMPACT.”
      • “… benefits were clinically confirmed by a significant reduction in infection rates and a reduction in wound healing complications, while average length of admission fell…” (Senkal et al. 1997)
      • “ Clinically, this group had a significantly shorter length of hospital stay, a lower sepsis score and a trend to decreased infection.” (Braga et al. 1996)
      • “ Complication rates and length of hospital stay were reduced…” (Daly et al. 1995)
      RNA CONTAINING FOODS CURRENTLY ON THE MARKET
      • SMA Baby Food
      • “… contains added nucleotides which have been shown to be involved in the development of an infant’s immune system and may also reduce the incidence of diarrhoeal disease in babies.”
      • Advanced Formula ENFAMIL
      • (Mead Johnson)
      • “ Nucleotide levels in ENFAMIL are patterned after the actual average free nucleotide levels found in breast milk.”
      RNA CONTAINING FOODS CURRENTLY ON THE MARKET
      • Breast milk has been reported to contain 1-12 mg/dL of DNA and 10-60 mg/dL of RNA.
      • Sanguansermsri J, 1974 Am. J. Clin. Nutr. 2: 859
      RNA CONTAINING FOODS CURRENTLY ON THE MARKET
      • Humans typically eat several grams of nucleotides in their diet each day.
    •  
    • LONGEVITY
      • “… research indicates that nucleotides such as RNA may help to reverse the effects of aging.”
      • “ One Swedish study found that supplemental RNA enhances the effects of antioxidants…”
      • “… many patients followed a [RNA] supplement regime for many years without exhibiting harmful side effects. Instead, they demonstrate many benefits, including diminished skin wrinkling, improved circulation and peripheral nerve function.”
      • “… rats injected weekly with RNA and DNA lived far longer than rats that did not receive the supplements.”
      • LE Magazine August 1997
    • LONGEVITY
      • “… efficacy of RNA was its ability to increase cellular energy levels so as to facilitate the movement of young cells to the surface where they could replace unsightly senescent cells.”
      • “ The nucleic acid RNA… seemed to restore this repair mechanism.”
      • LE Magazine January 2003
    • MEMORY
      • “ Other studies indicate that RNA may … play a role in helping … elderly people to avoid illness and remember things more clearly…”
      • LE Magazine August 1997
    • ENERGY
      • “… exogenous nucleosides increased the intercellular concentrations of UTP, UDP-glucose, CDP-choline and NAD + …”
      • Arnaud A, Exogenous nucleosides alter the intracellular nucleotide pool in hepatic cell cultures. Implications in cell proliferation and function. Clin Nutr, 2003 Aug; Vol. 22 (4), pp. 391-9
    • ENERGY
      • “ Nucleotide… food deprivation significantly decreased … the ATP pool…”
      • Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A, (1994) Dietary nucleotides accelerate intestinal recovery after food deprivation in old rats.Symposium: Nucleotides and Nutrition Supplement in J. Nutr. (WA Walker, ed.), vol. 124, pp1413-1418
    • BLOOD FLOW
      • “… the difference in blood flow velocities between baseline and 90 min was significantly greater with the formula with added nucleotides versus the formula with no added nucleotides… These data suggest that orally administered nucleotides are associated with affects on the intestinal vasculature.”
      • “ The higher … blood flow velocity measured after nucleotide-supplementation may reflect dilation of the intestinal vascular bed.”
      • Carver JD et al The Effects of Dietary Nucleotides on Intestinal Blood Flow Pediatric Research 52 (3) (2002)
      • “ Nucleotides … may decrease the inflammatory response to ischemia-reperfusion.” “… the number of leukocytes accumulated … was reduced in the presence of nucleotides. Furthermore, nucleotides may have decreased protein leak and the production of nitric oxide during ischemia.”
      • Bastamante, S. A., N. Sanches, J. Crosier, D. Miranda, G. Colombo, and M. J. S. Miller. 1994. Dietary nucleotides:effects on the gastrointestinal system in swine. J. Nutr. 124:149S-156S.
      BLOOD FLOW
    • BLOOD CELLS
      • “ Studies on the rat everted gut have found that the nucleotides inosine, hypoxanthine and uric acid all significantly increased iron absorption.”
      • Faelli A, Esposito G. Effect of inosine and its metabolites on intestinal iron absorption in the rat. Biochem Pharmacol 1970; 19: 2551-4.
    • DETOXIFICATION
      • “ Mycotoxin levels in the liver of pigs and poultry fed with nucleotide supplemented feed were significantly lower than of animals fed a standard diet, whereas the mycotoxin levels in the faeces of nucleotide fed animals were higher than of standard fed animals.”
      • Koppel P, Physiological and nutritional functions of nucleotides. Chemoforma LTD Switzerland. Glasgow Veterinary School February (2003.)
    • INFLAMMATION
      • “… peri-operative IMPACT* administration … reduced C-reactive protein release in the IMPACT group.”
      • *enteral formula containing nucleotides
      • O'Flaherty L, Immunonutrition and surgical practice. Proc Nutr Soc, 1999 Nov; Vol. 58 (4), pp. 831-7
    • INFLAMMATION
      • “… enhancement of IL-12 production is responsible for the augmentation of the Th1 immune response by dietary nucleotides … feeding of dietary nucleotides was found to decrease the antigen-specific IgE response significantly.”
      • Nagafuchi et al, Dietary nucleotides can up-regulate antigen specific TH1 immune responses in mice International Archives of Allergy and Immunology 122, 33-41 (2000)
    • ALLERGIC INFLAMMATION
      • “… report that mice fed nucleotide-supplemeted … diets had lower serum IgE levels, serum IgGI:IgG2a ratios and IL-4…”
      • “… dietary nucleotides may upregulate the Th1 immune response in systemic immunity.”
      • Nagafuchi S, Katayanag T, Nakagawa E, Takahashi T, Yajima T, Yonekubo A, et al. Effects of dietary nucleotides on serum antibody and splenic cytokine production in mice. Nutr Res 1997; 17: 1163-74
      • “ To date no side effects [of nucleotides] have been shown in clinical trials.”
      • Lerner A and R. Shamir. Nucleotides in Infant Nutrition: A Must or an Option
      • IMA. Vol 2, October 2000
      IMMUNE SURVELLIENCE SYSTEM
    • IMMUNE SURVELLIENCE SYSTEM
      • “ Mice maintained on a nucleotide-free diet have been found to exhibit deficient macrophage activity.”
      • Kulkarni AD, Fanslow WH, Drath DB, Rudolph FB, Van Buren Ct: Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice. Arch Surg 1986; 121: 169-172.
    • HUMORAL IMMUNE SYSTEM
      • “ Infants fed the nucleotide-supplemented formula had significantly higher serum… antibody concentrations…”
      • “… the nucleotide-supplemented formula was associated with higher antibody responses to two vaccines, Hib conjugate vaccine and DPT…”
      • Pickering L, Granoff D, Erickson JR, Masor M, Cordle CT, Scheller JP, Winship TR, Paule CL, Hilty MD. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics 1998;101:242–9.
      • Carver, JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatrica, 09/21/99, Vol. 88 Issue 9
      • “… using murine spleen cells showed that polynucleotides significantly increase in vitro antibody production in response to T-cell-dependent antigen. They seem to exert actions on T-helper cells at antigen presentation, perhaps during cognitive cell-cell interactions. … Polynucleotides increase in vitro human immunoglobulin production in response to T-cell-dependent stimuli and antigen. Humoral immune responses to T-cell-dependent antigen were depressed in mice fed a nucleotide-free diet, but were restored by a mononucleotide-nucleoside mixture.”
      • Jyonouchi H, (1994) Nucleotide Actions on the Humoral Immune Response
      • J. Nutr. 124, Suppl. January 1994: 138S -143S
      HUMORAL IMMUNE SYSTEM
      • “… higher plasma immunoglobulin M and A concentrations were found in preterm infants fed nucleotide-supplemented formula.”
      • Martinez-Augustin O, Boza J, Navarro J, Martinez-Valverde A, Araya M, Gil A. Dietary nucleotides may influence the humoral immunity in immunocompromised children Nutrition 1997; 13: 465-9 .
      HUMORAL IMMUNE SYSTEM
      • “ In mice and in humans, a nucleotide free diet significantly depressed IgM and IgG antibody production from spleen cells.”
      • Jyonouchi, H. (1994). Nucleotide actions on humoral immune responses. The journal of Nutrition. Nucleotides and nutrition. Vol 124. No 1S. 138S-143S.
      HUMORAL IMMUNE SYSTEM
      • “… the specific immunoglobulin A (IgA) level significantly increased in [mice] fed a nucleotide-supplemented diet compared with those fed a nucleotide-free control diet.”
      • Shinya N et al Dietary nucleotides increase mucosal IgA response and secretion of TGF Cytotechnology 40:49-58(2002 ).
      HUMORAL IMMUNE SYSTEM
      • “… oral nucleotides enhanced … the response to some T dependent protein antigens.”
      • Pickering L, Granoff D, Erickson JR, Masor M, Cordle CT, Scheller JP, Winship TR, Paule CL, Hilty MS. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics 1998; 101: 242-9 .
      CELLULAR IMMUNE SYSTEM
    • CELLULAR IMMUNE SYSTEM
      • “ In animal models, nucleotides have been shown to… improve delayed cutaneous hypersensitivity and alloantigen-induced lymphoproliferation…”
      • Pickering L, Granoff D, Erickson JR, Masor M, Cordle CT, Scheller JP, Winship TR, Paule CL, Hilty MD. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics 1998;101:242–9.
      • “… the effect of a nucleotide-free diet on the immune function of mouse syngeneic bone marrow radiation chimeras. … These results suggest that dietary nucleotides are important for the normal function of mouse T-lymphocytes.”
      • Kulkarni, S. S., D. C. Bhately, A. R. Zander, C. T. Van Buren, F. B. Rudolph, K. A. Dicke, and A. D. Kulkarni. 1984. Functional impairment of T-lymphocytes in mouse radiation chimeras by a nucleotide-free diet. Exp. Hematol. 12:694-699 .
      CELLULAR IMMUNE SYSTEM
      • Feeding nucleotide-supplemented … rodents has been associated with
      • increases in the following immune responses:
      • Graft-versus-host disease mortality;
      • Rejection of allogenic grafts;
      • Delayed cutaneous hypersensitivity;
      • Alloantigen- and mitogen-induced lymphoproliferation;
      • Reversal of malnutrition and starvation-induced immunosuppression;
      • Natural killer cell activity and macrophage activation;
      • Resistance to microbial challenge;
      • Macrophage phagocytic capacity;
      • Spleen cell production of interleukin-2 (IL-2) and expression of IL-2 receptors and lyt-1 surface markers;
      • The number of antibody-secreting spleen cells produced in cluture;
      • Popliteal lymp-node cytokine secretion;
      • Peripheral blood total leukocyte counts and neutrophil numbers following infection.
      • Carver, JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatrica, 09/21/99, Vol. 88 Issue 9
      IMMUNE SYSTEM
    • IMMUNE SYSTEM
      • “ Dietary nucleotides are reported to play a role in the maintenance of the immune response.”
      • Carver, JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatrica, 09/21/99, Vol. 88 Issue 9
    • IMMUNE SYSTEM
      • “ Spleen cells of mice maintained on a nucleotide-free diet produce less interleukin-2 (IL-2) and have lower natural killer (NK) cell cytotoxiicity and macrophage activiation compared with animals fed nucleotide-supplemented diet. In vivo lymphoproliferative response, macrophage phagocytic activity, and expression of IL-2 receptor and Lyt1 surface marker are also lower in animals fed nucleotide-free diet.”
      • Carver JD, Pimentel B, Cox WI, Barness LA. Dietary nucleotide effects upon immune function in infants. Pediatrics 1991;88:359–63.
    • “ RNA therapy shows signs of enhancing the immune system, improving transplanted organ viability and even fighting off cancers.” “… supplemental ribonucleic acid (RNA) may help boost our immune systems, increase energy levels, neutralize toxins, repair cell damage and improve skin elasticity.” LE Magazine August 1997 IMMUNE SYSTEM
    • “… studies suggesting potential benefits to intestinal flora, immunity, iron absorption, lipid metabolism and gut development.” Nucleotides in Infant Nutrition: A Must or an Option, Aaron Lerner and Raanan Shamir IMMUNE SYSTEM
    • IMMUNE HEALTH
      • “ In human, parenteral solutions with nucleotides given to postoperative cancer patients improved immune function and infectious complications and length of hospital stay was reduced compared to a control group.”
      • Van Buren, C.T., A.D. Kulkarni, F.B. Rudolph. (1994). The role of nucleotides in adult nutrition. The journal of Nutrition. Nucleotides and nutrition. Vol 124. No 1S. 160S-164S.
    • CANCER
      • “ Because dietary nucleotides are particularly essential for individuals under conditions of stress, oral administration of dietary nucleotides may result in tumor regression through enhancement of the production of IFN- γ and IL-12.”
      • Nagafuchi et al, Dietary nucleotides can up-regulate antigen specific TH1 immune responses in mice International Archives of Allergy and Immunology 122, 33-41 (2000)
    • CANCER
      • “… increased toxicity and tumor promotion observed in animals … may in part be due to resulting imbalances in dNTP pools and interference with DNA replication.”
      • Jackson C. et al Dietary Nucleotides:Effects on Cell Proliferation in Rats Fed Folate/Methyl-Deficient Diets. Journal of Nutrition 127(5) : 834S-837S.(1997)
    • INFECTION
      • “ [Anil D. Kulkarni and his colleagues] fed mice a diet supplemented with RNA and compared them with a control group of mice fed a nucleotide-free diet. Then they injected the mice with a strain of powerful bacteria. Half of the mice on the nucleotide-free diet died. The mice that had taken RNA, however, were able to fight off the infection.”
      • LE Magazine August 1997
    • INFECTION
      • “ There was an inhibition of gram-negative enteric bacteria … in mice fed … a diet supplemented with a nucleoside-nucleotide mixture… indicating that the nuclotide-nucleoside mixture blocked bacterial translocation by preventing endotoxin-induced mucosal or epithelial damage. These results suggest that the nucleoside-nucleotide mixture could be used ot inhibit or reduce the incidence of bacterial translocation, … and improve survival in a lethal model of … endotoxemia.”
      • Adjei AA, Yamamoto S. A dietary nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in mice fed protein-free diet. J Nutr 1995;125:42–8.
    • INFECTION
      • “ Mice on the nucleotide-free diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus while RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the nucleotide-free diet had decreased phagocytic activity compared with mice maintained on the nucleotide-free diet supplemented with adenine, uracil, or RNA. … provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge.”
      • Kulkarni AD, Fanslow WC, Drath DB, Rudolph FB and Van Buren CT, (1986a)
      • Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice Arch. Surg. 121: 169-172
    • INFECTION
      • “… dietary nucleotide restriction increases the mortality from staphylococcal sepsis. Addition of certain dietary nucleotides appears to decrease this susceptibility to bacterial challenge.”
      • Kulkarni AD et al Effect of Dietary nucleotides on response to bacterial infections. J Parenter Enteral Nutr. March 10(2):169-171 (1986)
    • INFECTION
      • “… mice maintained on a nucleotide-free diet exhibit a significantly decreased mean survival time and a significantly increased viable organism recovery in the spleen following intravenous injection of graded inocula of C. albicans compared to mice fed diets containing RNA or uracil as a nucleotide source.”
      • Fanslow WC, Kulkarni AD, Van Buren CT and Rudolph FB (1988) Effect of nucleotide restriction and supplementation on resistance to experimental murine candidiasis
      • J. Parenter. Enteral Nutr. 12: 49-52
    • HEART
      • “ An immune-boosting nutritional supplement can lower the risk of infection and death in patients undergoing heart surgery, researchers report. … high-risk patients who took a supplement containing … yeast RNA had lower rates of infection and organ dysfunction following coronary artery bypass than those who took an inactive placebo pill.”
      • Tepaske R, et al. Effect of preoperative oral immune-enhancing nutritional supplements on patients at high risk of infection after cardiac surgery: a randomised placebo-controlled trial. Lancet 358: 696-701(2001)
    • ICU PATIENTS
      • “ Early enteral feeding of the formula [containing dietary nucleotides] was safe and well tolerated in ICU patients. In patients who received the [nucleotide] formula, particularly if they were septic on admission to the study, a substantial reduction in hospital length of stay was observed, along with a significant reduction in the frequency of acquired infections.”
      • Bower RH, Cerra FB, Bershadsky B, Licari JJ, Hoyt DB, Jensen GL, Van Buren CT, Rothkopf MM, Daly JM, Adelsberg BR. Early enteral administration of a formula (Impact) supplemented with arginine, nucleotides, and fish oil in intensive care unit patients: results of a multicenter, prospective, randomized, clinical trial.
      • Crit Care Med. 1995 Mar;23(3):436-49 .
    • KIDNEY
      • “… beneficial effects support use of an oral immune-enhancing nutritional supplement in patients who are undergoing … surgery and are at high risk of infection. … The study included 50 patients who took the supplement or a placebo for at least 5 days before surgery. … About 17% of patients who took the supplement developed an infection, compared with 55% of those who took the placebo. Patients who took the supplement also had better kidney function and remained in the hospital for fewer days than patients who were given the placebo.”
      • Tepaske R, et al. Effect of preoperative oral immune-enhancing nutritional supplements on patients at high risk of infection after cardiac surgery: a randomised placebo-controlled trial. Lancet 358: 696-701(2001)
    • KIDNEY
      • “ Oral administration of nucleotides to fish daily resulted in enhanced … activities in kidney phagocytic cells. This activation of kidney cells was observed for at least 10 days post-treatment.”
      • Sakai, M; Taniguchi, K; Mamoto, K; Ogawa, H; Tabata, M. Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L. Journal of Fish Diseases, Oct2001, Vol. 24 Issue 8, p433, 6p
    • LIPID BALANCE
      • “ It has been reported that early childhood levels of serum lipids and lipoproteins are good predictors of lipid levels in adulthood.”
      • “… neonates fed formula supplemented with nucleotides from birth have significantly higher HDL-C and lower LDL-C serum levels than do neonates fed an unsupplemented formula.”
      • “… the only known difference between groups was the supplementation of formula with nucleotides. This factor was identified as one of the independent variables influencing the HDL-C and LDL-C levels in the multiple regression analysis models. Therefore, it can be assumes that dietary nucleotides have an effect on lipoprotein-cholesterol serum concentrations…”
      • Siahanidou. Serum Lipids in Preterm Infants Fed a Formula Supplemented With Nucleotides. Journal of Pediatric Gastroenterology & Nutrition, Jan2004, Vol. 38 Issue 1, p56, 5p
      • Webber LS, Srinivasan SR, Wattigney WA, et al. Tracking of serum lipids and lipoproteins from childhood to adulthood. Am J Epidemiol 1991; 133: 884-9
    • LIPID BALANCE
      • “… previous studies all conclude that formula supplementation with nucleotides influences circulating lipid levels…”
      • “ Sanchez et al. found higher HDL and lower VLDL plasma levels in supplemented neonates.”
      • “ The mechanism(s) by which dietary nucleotides affect HDL-C and LDL-C serum concentrations is/are not known.”
      • “ It has been suggested … that dietary nucleotides increase the synthesis of long-chain polyunsaturated fatty acids…”
      • Siahanidou. Serum Lipids in Preterm Infants Fed a Formula Supplemented With Nucleotides. Journal of Pediatric Gastroenterology & Nutrition, Jan2004, Vol. 38 Issue 1, p56, 5p
      • Sanchez-Pozo A, Pita ML, Martinez A, et al. Effects of dietary nucleotides upon lipoprotein pattern of newborn infants. Nutr Res 1986; 6: 763-71
    • LIPID BALANCE
      • “ It is well known that polyunsaturated fatty acids have a significant lowering effect on LDL-C circulating levels by increasing LDL receptor activity or reducing apoB secretion, and they also may increase the HDL lipoprotein levels by enhancing the apoA-1 secretion.”
      • “ The combination of high HDL-C and low LDL-C circulating levels is considered to be a protective factor against the development of vascular disease.”
      • Siahanidou. Serum Lipids in Preterm Infants Fed a Formula Supplemented With Nucleotides. Journal of Pediatric Gastroenterology & Nutrition, Jan2004, Vol. 38 Issue 1, p56, 5p
      • .
      • American Academy of Pediatrics. Committee on Nutrition. Cholesterol in Childhood. Pediatrics 1998; 101: 141-7.
    • LIPID METABOLISM
      • “ Dietary nucleotides … supplementation … had higher lecithin cholesterol acyl transferase (LCAT) activities and apoA-IV levels than those receiving the nucleotide-free formula. … These findings indicate that nucleotide supplementation … may improve dietary lipid tolerance by enhancing plasma LCAT activity, probably as a result of an increase in apoA-IV plasma concentrations…”
      • Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J, van Biervliet JP, Rosseneu M. Dietary nucleotides enhance plasma lecithin cholesterol acyl transferase activity and apolipoprotein A-IV concentration in preterm newborn infants. Pediatr Res 1995;37:328–33 .
    • LIPIDS
      • “… nucleotides are involved in the synthesis of proteins and other macromolecules such as phospholipids, and thereby facilitate lipoprotein synthesis. . . . These findings suggest that dietary nucleotides may enhance the synthesis of lipoproteins during the early neonatal period, especially in preterm infants.”
      • Sanchez-Pozo, A., J. Morillas, I. Molto, R. Robles, A. Gils. (1994). Dietary nucleotides influence lipoprotein metabolism in newborn infants. Pediatr. Res. 35. 112-116 .
    • STRESS
      • “… nucleotide-supplemented … groups shoed significantly lowered corticosterone levels, resulting in a decreased stress response… such a decrease in corticosterone levels may be one of the mechanisms of immune restoration. … Thus it is possible that RNA and uracil may have suppressed the production of this stress hormone and enhanced certain cytokines that resulted in the significant increase in immune response.”
      • Yamauchi K, Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog. J Appl Physiol, 2002 Jul; Vol. 93 (1), pp. 161-6
    • STRESS
      • “… under certain stress conditions … and extremely unusual environments including space flight, the endogenous supply of these compounds (nucleotides and nucleosides) may not be adequate for optimal functions…”
      • “ The results of our study have demonstrated that mice fed both CDR (RNA supplemented) and CDU (uracil supplemented) enhanced in vivo and ex vivo lymphocyte cell proliferation and IL-2 and INF-  production and decreased the stress level, indicated by corticosterone levels, compared with mice fed control CD (control diet).”
      • Yamauchi K, Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog. J Appl Physiol, 2002 Jul; Vol. 93 (1), pp. 161-6
    • STRESS
      • “ Roles for dietary sources of performed purines and pyrimidines such as RNA in various systems have been demonstrated in recent years. … These compounds were previously thought to be non-essential but are now considered to be conditionally requires when various stresses … are present.”
      • Rudolph FB. The metabolic effects of enterally administered ribonucleic acids. Curr Opin Clin Nutr Metab Care, 1998 Nov; Vol. 1 (6), pp. 527-30
    • STRESS
      • “ The effect of dietary nucleotide restriction was tested during … stress… These studies … clearly indicate the nutritional role of nucleotides…”
      • Pizzini R, Kumar S, Kulkarni Ad, Rudolph FB and Van Buren CT, (1990)
      • Dietary nucleotides reverse malnutrition and starvation-induced immunosuppression
      • Arch. Surg. 125: 86-90
    • ULCERS
      • “ We conclude that the yeast RNA … accelerated ulcer healing, as indicated by decreased ulcer number and length. We postulate that the underlying mechanism(s) contributing to ulcer healing may be related, in part, to increased cell proliferation.”
      • Sukumar P, Loo A, Magur E, Nandi J, Oler A, Levine RA. Dietary supplementation of nucleotides and arginine promotes healing of small bowel ulcers in experimental ulcerative ileitis. Dig Dis Sci. 1997 Jul;42(7):1530-6.
    • ULCERS
      • “… enteral and parenteral supplementation of nucleotides accelerates healing of … ulcers in rats…”
      • Sukumar et al Dietary Nucleotides Augment Distal Colitis in Rats Journal of Nutrition 129:1377-1381 (1999).
    • STOMACH
      • “… dietary nucleotides are reported to play a role in growth and differentation of the gastrointestinal tract.”
      • Uauy R. Dietary nucleotides and requirements in early life. In: Lebenthal E, editor. Textbook of gastroenterology and nutrition. New York: Raven Press, 1989: 265-80
      • “ Maltase activity was significantly greater … the largest increase, 87%, was seen in the proximal gut mucosa. The maltase/lactase ratio was also higher in this segment.”
      • Uauy, R. G. Stringel, R. Thomas, and R. Quan. 1990. Effect of dietary nucleotides on growth and maturation of the developing gut in the rat. J. Pediatr. Gastroenterol. Nutr. 10:497-503 .
      ENZYMES
    • ENZYMES
      • “ Nucleotide… food deprivation significantly decreased … maltase, sucrase, lactase and aminopeptidase activities.”
      • Ortega MM, Nunez MC, Gil A and Sanchez-Pozo A, (1994) Dietary nucleotides accelerate intestinal recovery after food deprivation in old rats.Symposium: Nucleotides and Nutrition Supplement in J. Nutr. (WA Walker, ed.), vol. 124, pp1413-1418
    • INTESTINAL TRACT
      • “… investigators report decreased protein synthesis in the small intestine of rats fed nucleotide-free diets and accelerated intestinal recovery following food deprivation in rats fed nucleotide-supplemented diets. Dietary nucleotides may also be beneficial following intestinal injury.”
      • “… intestinal histology and ultrastructure were improved by feeding nucleotide-supplemented versus nucleotide-free diets to rats following chronic diarrhea.”
      • “ Dietary nucleotide supplementation promoted healing of small bowel ulcers in experimental ulcerative ileitis in rats…”
      • Carver, JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatrica, 09/21/99, Vol. 88 Issue 9
    • INTESTINAL TRACT
      • “ Infants receiving the supplemented formula experienced fewer first episodes of diarrhea…”
      • “… infants fed nucleotide-supplemented formula had a significantly lower incidence of diarrhea compared with infants fed unsupplemented formula.”
      • Carver, JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatrica, 09/21/99, Vol. 88 Issue 9
    • INTESTINES
      • “… studies have shown that rapidly dividing cells of human intestinal tract are dependent on dietary nucleotides for their replication.”
      • Sanderson, I.R., H. Youping. (1994). Nucleotide uptake and metabolism by intestinal epithelial cells. The journal of Nutrition. Nucleotides and nutrition. Vol 124. No 1S. 131S-137S.
    • DIARRHEA
      • “ The effects of a nucleotide-supplemented formula on diarrhoeal disease was studied… [The supplemented group] experienced less episodes of diarrhoea including less first episodes and for a lesser number of days … and [a larger proportion] never developed episodes of diarrhoea. … The mechanisms through which nucleotides decrease the incidence of diarrhoeal disease in infants remain unclear.”
      • Brunser O, Espinoza J, Araya M, Cruchet S, Gil A. Effect of dietary nucleotide supplementation on diarrhoeal disease in infants. Acta Paediatr 1994;83:188–91 .
    • DIARRHEA
      • “… the incidence and duration of acute diarrhoea is lower in infants fed supplemented-nucleotide formulas.”
      • Gil A Modulation of the immune response mediated by dietary nucleotides. Eur J Clin Nutrition August 56(3):S1-4 (2002)
      • “… results suggest that dietary nucleotides promote earlier restoration of the ileal mitochondrial function after chronic diarrhea.”
      • Arnaud, Journal of Pediatric Gastroenterology & Nutrition; Aug2003, Vol. 37 Issue 2, p124, 8p
      DIARRHEA
    • LIVER
      • “… aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamic acid- oxalacetic transaminase (GOT) and glutamic acid- pyruvic transminase (GPT) levels were significantly lower in animals given a nucleotide-supplemented diet than those given the standard nutrition, indicating faster recovery from liver injury.”
      • Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y, Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects of total parenteral nutrition with nucleotide and nucleoside mixture on D-galacosamine-induced liver injury in rats.
      • J. Paranter. Enter. Nutr. 12: 53-57
    • LIVER
      • “ Parenterally administered nucleotides promote recovery of liver injury … and improve liver function and nitrogen balance after liver injury or partial hepatectomy.”
      • Ogoshi, S., Iwasa, M., Kitagawa, S., Ohmori, Y., Mizobuchi, S., Iwasa, Y. & Tamiya, T. (1988) Effects of total parenteral nutrition with nucleosides and nucleotide mixture on D-galactosamine-induced live injury in rats. J. Parenter. Enteral Nutr. 18: 62-66.
    • LIVER
      • “ In pre-term infants nucleotide supplement may improve lipid tolerance by enhancing plasma lecithin cholesterol acyltransferase activity.”
      • Sanchez-Pozo A, Ramirez M, Gil A, Maldonado J, van Biervliet JP, Rosseneu M. Dietary nucleotides enhance plasma lecithin cholesterol acyltransferase activity and apolipoprotein A-IV concentration in preterm newborn infants. Pediatr Res 1995; 37: 328-33.
    • LIVER
      • “ The use of dietary nucleotides … was found to decrease the percentage area of fibrous septae. In animals with liver cirrhosis fed the nucleotide-supplemented diet for two weeks, the total area of fibrosis was reduced. … In conclusion, dietary nucleotides may be an important factor in the histological recovery of damaged liver in experimental cirrhosis.”
      • Torres M. I. Effect of Dietary Nucleotides on Degree of Fibrosis and Steatosis Digestive Diseases and Sciences , 42 (6) :1322-132.(1997) .
    • LIVER
      • “ The serum GOT and GPT concentrations were significantly lower in the group supplemented with nucleoside-nucleotide mixture than those in other groups.”
      • “… the nucleoside/nucleotide mixture … inhibited the [liver] injury. … Thus infusion of a physiological and balanced mixture of nucleosides or nucleotides may improve liver function in rats with liver injury.”
      • Ogoshi S, Iwasa M, Kitagawa S, Ohmori Y, Mizobuchi S, Iwasa Y and Tamiya T, (1988) Effects of total parenteral nutrition with nucleotide and nucleoside mixture on D-galacosamine-induced liver injury in rats. J. Paranter. Enter. Nutr. 12: 53-57
    • BRAIN
      • “ The learning ability of rats fed the nucleotides-supplemented diet, which was evaluated by the water-filled multiple T-maze test and passive avoidance test, was superior to the rats fed the nucleotides-free diet. The results presented here suggest that dietary nucleotides may influence lipid metabolism of the cerebral cortex and contribute to the rise in learning ability of rats.”
      • Sato N, Effects of dietary nucleotides on lipid metabolism and learning ability of rats. Biosci Biotechnol Biochem, 1995 Jul; Vol. 59 (7), pp. 1267-71
    • FATTY ACIDS
      • “… dietary nucleotides play a role in the in vivo desaturation and elongation of essential fatty acids to long chain PUFA during early life for the human newborn.”
      • DeLucchi C, Pita ML, Faus MJ, Molina JA, Uauy R, Gil A. Effects of dietary nucleotides on the fatty acid composition of erythrocyte membrane lipids in term infants. J Pediatr Gastroenterol Nutr 1987;6:568–74.
    • FATTY ACIDS
      • “ Plasma polyunsaturated fatty acids with more than 18 carbons of the omega 6 family were significantly increased… These results suggest that dietary nucleotides may be involved in the conversion of linoleic acid to longer chain polyunsaturated fatty acids during early life.”
      • Gil A, Pita M, Martinez J, Molina JA and Sanchez Medina F, (1985)
      • Effect of dietary nucleotides on the plasma fatty acids in at-term neonates.
      • Hum. Nutr. Clin. Nutr. 40: 185-195
    • PROTEIN SYNTHESIS
      • “ These findings suggest that the nucleoside-nucleotide mixture was used for the syntheses of RNA and DNA through the salvage pathway … and that this resulted in enhanced protein turnover with simultaneous increase in protein synthesis.”
      • Ogoshi S, Iwasa M, Yonezawa T and Tamiya T, (1985) Effect of nucleotide and nucleoside mixture on rats given total parenteral nutrition after 70% hepatectomy.
      • J. Paranter. Enter. Nutr. 9: 339-342
    • APOPTOSIS
      • “… in vitro modulation of cell proliferation and apoptosis has been described by ribonucleosides, in particular by modified components using human cell culture models.”
      • Schlimme, E., D. Martin, and H. Meisel. 2000. Nucleosides and nucleotides: natural bioactive substances in milk and colostrums. Br. J. Nutr. 84:S59
    • CYTOKINES
      • “ Dietary nucleotides have been shown to enhance the production and the genetic expression of IL-6 and IL-8… Dietary nucleotides may … affect the levels of intestinal cytokines.”
      • Gil A Modulation of the immune response mediated by dietary nucleotides. Eur J Clin Nutrition August 56(3):S1-4 (2002)
    • NERVES
      • “ Guanine nucleotides regulate the affinity of melatonin receptors… The effect of guanine nucleotides and related analogues on the binding of melatonin to membranes … was studied. … These results are consistent with a melatonin receptor existing in an equilibrium between high- and low-affinity states, with GTP and related analogues able to cause a shift in the equilibrium…”
      • Morgan P.J.et al . Guanine nucleotides regulate the affinity of melatonin receptors on the ovine pars tuberalis. Neuroendocrinology, 50, 359362. (1989)
    • SKIN
      • “… topical application of RNA improved cell energy metabolism and therefore the health and appearance of the skin.”
      • “ Dietary nucleotides affect … tissue repair.”
      • LE Magazine January 2003
      • Ramírez M, Effect of dietary nucleotides and orotate on the blood levels of prostacyclin (PGI2) and thromboxane (TXA2) in the weanling rat. Prostaglandins Leukot Essent Fatty Acids, 1991 May; Vol. 43 (1), pp. 49-54
      • “ In animal models, nucleotides have been shown to… enhance T-cell maturation and function.”
      • Pickering L, Granoff D, Erickson JR, Masor M, Cordle CT, Scheller JP, Winship TR, Paule CL, Hilty MD. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics 1998;101:242–9.
      MICROBES
    • MICROBES
      • “ One possible anti-infective mode of action of nucleotide may be that dietary nucleotide can inhibit endotoxin-induced bacterial translocation in protein-malnourished mice.”
      • Adjei AA, Yamamoto S. A dietary nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in mice fed protein-free diet. J Nutr 1995; 125: 42-8.
    • WEIGHT LOSS
      • “ Dietary RNA is required to restore lost immune function after protein deprivation. Adequate calories and protein alone do not return immune function to normal. Dietary nucleotides can restore lost immune function even during protein starvation and weight loss.”
      • Van Buren C.T., Kulkarni A.D.. Rudolph F.B., The Role of Dietary Nucleotides in Adult Nutrition, The Journal of Nutrition, January; 124:124S-160S.(1994)
    •  
    •  
    • RNAi: What's All the Noise About Gene Silencing? Posted 04/02/2004 Ernie Hood Introduction It all began with petunias. In the late 1980s, geneticist Richard Jorgensen, then working at a California plant biotechnology company, attempted to deepen the hue of purple petunias by introducing more of the gene that gives them their color, in the form of double-stranded RNA (dsRNA). Instead, some of the engineered flowers became variegated and others turned white, indicating that expression of both the introduced pigmentation gene and its homologous endogenous gene had been knocked down or knocked out altogether. Jorgensen had serendipitously discovered an age-old natural biologic process now recognized to be evolutionarily conserved in most, if not all, forms of life. Today, gene silencing—or RNA interference (RNAi), as it is now known—has revolutionized genetics and is on the verge of spawning an entirelynew class of drugs to treat human diseases with a genetic component. The ability to selectively silence genes is one of the hottest topics in biology today. Science crowned RNAi as its "Breakthrough of the Year" in 2002. Nobel laureate and RNAi pioneer Phillip Sharp, who is Salvador E. Luria Professor of Biology and director of the McGovern Institute for Brain Research at the Massachusetts Institute of Technology (MIT), calls it "the most exciting discovery in the last decade," adding that "there's not an area of biological science this will not touch." John Maraganore, who is president, CEO, and director of Alnylam Pharmaceuticals, touts RNAi as "presenting perhaps the broadest new class of therapeutics since recombinant proteins and monoclonal antibodies." Can RNAi live up to the hype? That remains to be seen, of course, but academic and industrial researchers are optimistic that it can and will, if the significant remaining barriers to its progress can be overcome. Given the rapid pace of discovery in the field, such optimism may well be justified. From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences
    • Cont. RNA Redefined It once seemed so simple, so straightforward: basically, DNA makes messenger RNA (mRNA); mRNA makes proteins. But the discoveries associated with RNAi have shown that the real story is far more complex. RNA has been unveiled as the "man behind the curtain" in the cell, wielding previously unimagined control over and influence upon cellular processes (including gene expression and regulation) and organism development. RNAi has been revealed to be an ancient mechanism protecting cells from invading viruses and from damage by transposable genetic elements, performing a variety of cellular housekeeping functions essential to survival, health, and development. RNAi was first described and so named by molecular biologists Andrew Fire of the Carnegie Institute of Washington and Craig Mello of the University of Massachusetts, along with their colleagues, in a landmark 19 February 1998 Nature paper that electrified the biology community. The team found that administering tiny amounts of dsRNA to Caenorhabditis elegans resulted in potent sequence-specific gene silencing. Tantalized by the possibility of acquiring a powerful new tool for genetic manipulation and analysis, investigators around the world began investigating RNAi. The flood of significant discoveries that followed soon established the basic outlines of the mechanisms involved in RNAi. Researchers using Drosophila found in 2000 that long-strand dsRNA was processed in cells into 21- to 23-nucleotide snippets of RNA, which then cleaved to precisely matching homologous mRNA sequences, degrading the mRNA and effectively silencing the corresponding gene by blocking its ability to encode for proteins. The higher life forms, such as mammals, while conserving this ability, use it in different ways; the response to dsRNA is more complicated, triggering a cellular immune response involving the release of interferon that ultimately kills the cell. From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. RNA Redefined Then, in 2001, Thomas Tuschl, then of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, discovered with his colleagues that RNAi could be prompted through the use of shorter pieces of RNA known as small interfering RNAs (siRNAs). Soon thereafter, they showed that duplexes of 21-nucleotide siRNAs mediated RNAi in cultured mammalian cells and demonstrated that siRNAs could be designed to silence specific genes without activating the interferon response. In other words, scientists could potentially silence any gene of interest in a highly predictable, reproducible, and accurate fashion. Research scientist Gregory Hannon and his colleagues at New York's Cold Spring Harbor Laboratory contributed several key discoveries during the same period. They identified, described, and named the "Dicer" enzyme, which chops dsRNA into siRNAs, as well as the RNA-induced silencing complex (RISC), which mediates the silencing process by degrading the homologous mRNA. In 2002, they described the use in mammalian cells of so-called short hairpin RNAs (shRNAs), which generate endogenous siRNAs within cells and thus provide stable, heritable gene silencing (in contrast, administered siRNAs are transient in their silencing effect). They whimsically named this effect "short hairpin-activated gene silencing," or SHAGging. This discovery allowed the development of cell lines and animal models with permanently silenced genes—a major step forward for basic science in general, and especially for functional genomics. Further advances in the past few years have added the ability to silence the expression of just the mutant copy of a gene, leaving the normal copy intact, as well as to modulate the level of silencing in order to produce a range of phenotypes. Plus, researchers can now induce silencing in a controlled manner and target multiple genes for silencing. These discoveries alone are quite important—all of these capabilities are crucial in a variety of critical applications. But some proponents believe this is only the beginning, and the best may be yet to come.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. Knock Down Genes, Drag Out Knowledge With the continual refinement and improvement of techniques to silence genes with exquisite specificity, RNAi has already had a major impact on molecular biology. For example, the pace of discovery in functional genomics has accelerated as a consequence of researchers' enhanced ability to practice reverse genetics, in which a gene's function can be inferred by silencing its expression. With complete sequences of several genomes now on hand, including those of C. elegans , Drosophila , the mouse, and the human, investigators can now quickly, easily, reliably, and relatively inexpensively use siRNAs to silence genes of interest and determine their functions. Several companies are already selling made-to-order siRNAs for use in functional genomics work, as well as for drug target identification and validation. The ability to knock down genes either stably (that is, creating heritable phenotypes through germline transmission of permanently silenced genes) or transiently (as opposed to knocking them out altogether) has some important advantages in the production of animal models and in vitro cell lines. When a gene is considered silenced by RNAi, expression is typically reduced by 70% or more. This allows the method to be used in so-called essential genes, which cannot be knocked out in animal models without killing the animal. Also, "turning down" a gene by a certain amount can sometimes more closely resemble a disease state, allowing the fashioning of more useful, refined models of some diseases. Russell Thomas, director of the functional genomics research program at the CIIT Centers for Health Research in Research Triangle Park, North Carolina, points out another advance that RNAi has brought to animal studies: "When you knock out a gene in mice, you have to live with the consequences," he says. "The targeted gene is knocked out for the remainder of the animal's life span. In contrast, shRNAs with inducible promoters allow an investigator to control the timing for the knockdown of the targeted gene. With this technology, you don't have residual developmental effects, and you can have more sophisticated experimental designs since you can look at wild-type expression and knockdown in the same animal and at multiple times throughout the animal's life span."
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. Knock Down Genes, Drag Out Knowledge Sharp observes that RNAi will in some cases obviate the need for animal knockouts. "You can do gene knockouts in mice, and then relate phenotype to somatic cells in the human, but it's very expensive, the gene has to be nonessential to get an interesting phenotype, and you have to ask the question in the context of the developing mouse," he says. By comparison, he explains, RNAi allows researchers to inactivate the gene and observe in real time the changes in the metabolic, cell biologic, or other phenotype of the cell, and characterize the role of the gene in that particular situation. Although these applications of RNAi are not yet perfected and are unlikely to completely replace classic knockout studies, many scientists are excited about adding these new capabilities and efficiencies to their bag of laboratory tricks. "It's fundamentally changing how we do laboratory science," says Sharp. "It will change how we do animal genetics, and we have not even scratched the surface of all the ways it will be used.“ RNAi and the Big Picture The ability to reproducibly and robustly silence every single gene in the genome is expected to facilitate the acquisition of profound new knowledge regarding function and regulation at the cell and whole-organism levels. Several organizations are in the process of constructing large-scale RNAi libraries that should be available for use very soon. Genomewide screening using RNAi libraries will help researchers learn more about global questions in systems biology, elucidating the nature and role of the complex, often interrelated pathways and signaling networks at work in organisms.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. RNAi and the Big Picture Leona Samson, director of the MIT Center for Environmental Health Sciences, has already used such a library for the 4,800 nonessential genes in Saccharomyces cerevisiae . She says that higher-organism RNAi construct libraries will aid toxicogenomics research by allowing researchers to interrogate each and every gene of an organism—to examine a specific gene to determine its function, or to screen large amounts of genes in the context of a specific function, to see which ones contribute to the function, and possibly what roles they might play and how they might interact. As a result, Samson says, "you can start to identify the important pathways for helping cells recover from toxic insults.“ Samson says there are important toxicogenomics treatment and prevention end points to be achieved with this type of large-scale screening. By interrogating every gene, it's possible to compile a portrait of all the pathways that are relevant to cellular function and response, such that researchers could look at which genes are being expressed in members of a population and accurately predict the effects of exposure to particular environmental agents. "In the end," Samson says, "we're going to get an integrated systems view, and we need that to be able to make predictions.“ There's More to the Machinery It turns out that gene silencing through degradation of mRNA by siRNAs is not the only cellular mechanism regulated by small pieces of RNA. Thanks to groundbreaking work in C. elegans , researchers have discovered a class of natural small RNA molecules called microRNAs that appear to be crucial in regulating development. Although they apparently use the same tools as siRNAs to carry out their functions—Dicer, RISC, and a family of proteins known as Argonautes—microRNAs differ in that their sequences do not precisely match their mRNA targets. As such, they regulate the expression of proteins by those targets, rather than degrade them altogether, which leads investigators to believe that they play an important role in the timing and nature of development—perhaps to the point of controlling differentiation in embryonic stem cells.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. There's More to the Machinery Tuschl, who is now group leader of the Laboratory of RNA Molecular Biology at Rockefeller University, says, "It's a very complicated regulatory machinery that you have in your cells, with a very complicated biology behind it. We know that there are genes that express dsRNA and microRNAs, and that this gene family in humans is about 250 genes, and many of these genes are conserved. . . . The question is what all these genes are doing, and how the RNAi machinery ties in to the gene regulation mediated by the microRNA genes." Recent studies further suggest there is yet a third mechanism controlled by microRNAs, an arm of the silencing machinery in the nucleus of the cell that modifies heterochromatin. The result is transcriptional repression of gene expression. As Tuschl puts it, "This is one of the real highlights of the discovery of RNAi—that it's a new cellular mechanism involved in regulating gene expression, and it's as complicated and as effective as transcriptional gene regulation." More thorough understanding of this mechanism could eventually lead to beneficial insights into development and disease processes, particularly carcinogenesis. Silencing Disease Although biologists are excited about the long-range additions to knowledge that could emerge from studying the complexities of the RNAi machinery, many are focusing major efforts on exploiting what is already known—that the gene-silencing effect of RNAi holds tremendous promise in treating human disease. RNAi therapeutics will be judged on the same criteria as any other prospective drug: potency, stability, and safety. Despite the great deal of work yet to be done, many researchers believe that RNAi-based agents will eventually pass muster on those issues, and will actually have inherent advantages over presently available classes of drugs.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. Silencing Disease For example, the fact that RNAi is a natural cellular process may mean that drugs based upon the phenomenon can be expected to be quite efficacious. "The siRNA process depends upon the endogenous pathway of microRNA biology, a process that's present in all cells," says Sharp. "Therefore, it's efficient, it's a normal biological process, and we are learning how to design siRNAs that are more efficiently taken up in that process and more efficiently used in silencing.“ Maraganore agrees. "What's very unique about RNAi is that it's the first natural mechanism that's ever been discovered that would allow people to silence genes," he says. "Because of that leveraging of that natural mechanism, you have a high degree of specificity and potency in the action of siRNAs." According to Nassim Usman, chief operating officer and senior vice president of Sirna Therapeutics in Boulder, Colorado, there is ample precedent for this opinion. "If you look at the history of successful biotechnology drugs, a lot of them that have been successful are the ones that either use or replace a naturally occurring mechanism," he says. "The two clearest examples are recombinant proteins and antibodies.“ The potential therapeutic value of RNAi has been repeatedly demonstrated in a wide variety of in vitro studies, and more and more in vivo experiments are confirming that early promise. Efficacy in gene silencing has been shown in viral diseases (such as HIV/AIDS, influenza, human papillomavirus infection, various hepatitis strains, smallpox, and SARS), neurodegenerative diseases (such as Parkinson disease, amyotrophic lateral sclerosis, and Alzheimer disease), cancer, inflammatory diseases (such as rheumatoid arthritis), and autoimmune diseases (such as type 1 diabetes mellitus). The intense interest in RNAi therapeutics has come at least in part because of its potential broad applicability across such a wide spectrum of disorders. Researchers also cite the specificity of RNAi targeting: siRNAs may be able to effectively reach cellular targets that have previously been inaccessible or highly resistant to other forms of therapy. Success in reaching such targets could lead to significant advances in the treatment of several diseases with currently unmet medical needs.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. The Principle of the Thing Researchers in academic and industrial labs around the world are pursuing therapeutic applications of RNAi. One good illustration is the work conducted by senior investigator and associate pediatrics professor Judy Lieberman and her group at the Institute for Biomedical Research of the Harvard Medical School Center for Blood Research. In 2003, the team published two RNAi studies of landmark importance. In one, an in vitro experiment presented in the Journal of Virology in July 2003 (issue 13), the team achieved sustained siRNA-mediated silencing of HIV-1 in primary macrophages, in essence preventing infection from taking hold. In previous studies conducted in rapidly dividing cells, such as tumors, the RNAi effect lasted only 3-5 days. In these macrophages, however, as Lieberman explains, "the silencing lasted very long, in fact for as long as we could keep the cultures growing, for some genes." The experiment co-targeted the viral p24 structural gene and CCR5, the major HIV-1 co-receptor in macrophages, cells that are known to be reservoirs of HIV infection and that are stubbornly resistant to current antiviral therapies. The team found that they could completely suppress HIV replication in macrophages by using this co-targeting stratagem. They also found that they could inhibit viral replication in cells that were already infected, in which HIV was integrated into the host cell. As Lieberman summarized, those are "pretty encouraging data for the possibility of using siRNA against HIV." The experiment also showed that RNAi holds promise as an antiviral treatment by silencing both host cell receptors and viral replication genes. In the other major study, published in the March 2003 issue of Nature Medicine , Lieberman and her colleagues demonstrated that RNAi could effectively treat or prevent disease in vivo . They successfully prevented liver failure and fibrosis in two mouse models of autoimmune hepatitis by silencing the Fas gene, which encodes the Fas receptor. Many liver diseases are characterized by apoptosis, which is mediated by the Fas protein.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. The Principle of the Thing "It turns out that the liver damage in a lot of kinds of hepatitis—even environmentally caused hepatitis, such as from alcohol or carbon tetrachloride exposure—all goes through the same final pathway," says Lieberman. "Hepatitis B and C are not pathogenic viruses. It's really the immune cells that infiltrate into the liver that become activated, engage the receptor that's expressed on liver cells, and trigger death." The group of mice treated with the Fas siRNA were protected when challenged with a hepatitis-inducing agent, and in a related experiment, most of the animals in the treated group were cured after having been subjected to a particularly aggressive model of hepatitis. "The results were pretty dramatic," says Lieberman, "and they were obtained without any optimization." With these and similar results reported by other investigators, it's not surprising that hepatitis is one of the initial treatment targets being pursued by RNAi biotechnology companies. With a $12 million grant from the National Institute of Allergy and Infectious Diseases, Lieberman's group, along with colleagues at MIT, is also studying the potential application of RNAi as a weapon against bioterrorism. The four projects funded by the grant include one led by Sharp to probe how RNAi and viral infection interact, another led by Lieberman designed to look at delivery questions, a third led by MIT assistant immunology professor Luk van Parijs looking at the effects of silencing various immune genes, and a fourth led by Harvard assistant pediatrics professor Premlata Shankar looking specifically at the possible application of RNAi against viral bioterrorism agents such as flaviviruses and poxviruses. Successful proof-of-principle studies are a long way from proven clinical safety and efficacy, of course, but academic and industrial researchers alike are confident that RNAi therapies will move rapidly from bench to bedside. Usman says Sirna expects to file an investigational new drug application with the U.S. Food and Drug Administration for an siRNA-based drug for age-related macular degeneration later this year. That would be a remarkable milestone, considering that the RNAi phenomenon itself has only been recognized for six years.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. The Principle of the Thing The rapid pace of discovery and development of RNAi therapeutics should continue, according to Maraganore. "It's a technology that, while early in terms of its discovery, lends itself to a very rapid cycle from target identification to lead optimization all the way through the start of formal investigational new drug-enabling studies," he says. Delivery, Delivery, Delivery Delivery—getting those exquisitely specific siRNAs or shRNAs to the appropriate sites in the appropriate amounts to ensure appropriate uptake and the intended silencing—remains a considerable challenge. Experts in the field agree that delivery is a daunting barrier to successful RNAi therapy. However, RNAi biotech companies and their backers are banking on overcoming the delivery barrier, and academic researchers seem confident that it can be done. As Tuschl explains, there are basically two strategies for delivering siRNAs in vivo . One strategy is gene therapy, which uses a viral vector to deliver the siRNA to the cells of interest. The other route is the chemical synthesis of the reagent, using some chemical modifications that change the properties of the siRNA such that they are more stable and are retained longer in the bloodstream; this simultaneously changes their uptake properties and allows more opportunity for uptake. With the broad applicability of RNAi to a diverse range of human diseases in a wide variety of organ systems, both delivery methods are being pursued for specific therapeutic targets. As RNAi therapies make their way into the clinic, it is perhaps inevitable that a more traditional dichotomy will emerge in delivery: local versus systemic administration. Some organs are simply much easier than others to reach with drugs. Says Maraganore, "The delivery hurdles are going to be more significant for systemic uses of RNAi—in other words, administration of siRNAs either intravenously or subcutaneously—as compared to an approach we call direct RNAi, which is the application of siRNAs to certain anatomical sites, for example the eye or the central nervous system."
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. Delivery, Delivery, Delivery With direct RNAi, drugs can perform their actions at such sites without having to negotiate the gastrointestinal tract or other hurdles that must be faced to reach less accessible organs. Maraganore believes direct RNAi will be the first approach to yield candidates that are ultimately approved, followed shortly thereafter by approaches that use systemic administration. The eye has been one of the first targets of siRNA therapeutics in development. Local delivery of an siRNA to the eye via intravitreal injection or topical administration is aimed at controlling the proliferation of abnormal blood vessels associated with one form of age-related macular degeneration. Several RNAi biotech companies are working on siRNAs designed to block the vascular endothelial growth factor pathway, a validated target of therapy in this disease. With the liver being another relatively easy drug delivery site, other companies are also in hot pursuit of an siRNA candidate compound to treat hepatitis C. Alnylam, meanwhile, is collaborating with the Mayo Clinic to develop an siRNA targeting the gene that encodes for β-synuclein, a protein recently discovered by the Mayo Clinic and the NIH to be overexpressed in people with Parkinson disease and thought to be a causative factor in the disease. The RNAi therapy would be delivered to the central nervous system by catheterization, another local delivery option in certain situations. In terms of systemic delivery of RNAi therapeutic agents, some researchers believe DNA-based vectors will be the way to go. Australia's Benitec, for example, has developed a technique it has dubbed "DNA-directed RNAi," which it claims allows for the inducible transient or permanent silencing of multiple genes. That approach could prove beneficial in the treatment of diseases such as HIV/AIDS and cancer, in which combination therapy attacking multiple targets simultaneously is an accepted therapeutic stratagem. Given the spotty track record and regulatory scrutiny of gene therapy, however, other developers are hitching their wagons to the refinement of siRNAs themselves as drugs.
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. Caution Signs Optimism about the vast potential of RNAi must, of course, be mitigated by the appropriate scientific reserve. At least some of the delivery hurdles could yet prove to be intractable. There could be unanticipated off-target effects, in which an siRNA knocks down unintended genes, potentially provoking a toxic release of interferon. However, experts such as Lieberman, while recognizing that "things in biology have a way of becoming more complicated than anyone can foresee," seem confident that obstacles in RNAi's path to the clinic can be overcome. "In the last six months," says Lieberman, "some of the most optimistic views of RNAi—that it was completely specific, that it wouldn't activate interferon—have turned out to not be absolutely true. But I still don't think that they're going to be serious problems in vivo ." Drug discovery and development experts point out that phenomena that might raise safety concerns in vitro often do not turn out to be clinically significant. Today, RNAi is already an accepted and vital tool of the science trade, yielding important new knowledge day in and day out. In medicine, RNAi is still in its infancy, but is rising just over the horizon as one of the first tangible and widespread benefits to be derived from the sequencing of the human genome. Aside from the potential of RNAi to benefit human health, scientists are also looking at the prospective ability of the technology to silence genes in economically important plant and animal species. For example, Japanese researchers reported in the 19 June 2003 issue of Nature the successful construction of transgenic coffee plants that are naturally decaffeinated by knocking down a gene involved in caffeine biosynthesis. Now if they could just do something about those washed-out petunias . . .
    • From Environmental Health Perspectives 112(4):A225-A229, 2004. National Institute of Environmental Health Sciences Cont. RNAi and the Big Picture Leona Samson, director of the MIT Center for Environmental Health Sciences, has already used such a library for the 4,800 nonessential genes in Saccharomyces cerevisiae . She says that higher-organism RNAi construct libraries will aid toxicogenomics research by allowing researchers to interrogate each and every gene of an organism—to examine a specific gene to determine its function, or to screen large amounts of genes in the context of a specific function, to see which ones contribute to the function, and possibly what roles they might play and how they might interact. As a result, Samson says, "you can start to identify the important pathways for helping cells recover from toxic insults.“ Samson says there are important toxicogenomics treatment and prevention end points to be achieved with this type of large-scale screening. By interrogating every gene, it's possible to compile a portrait of all the pathways that are relevant to cellular function and response, such that researchers could look at which genes are being expressed in members of a population and accurately predict the effects of exposure to particular environmental agents. "In the end," Samson says, "we're going to get an integrated systems view, and we need that to be able to make predictions.“ There's More to the Machinery It turns out that gene silencing through degradation of mRNA by siRNAs is not the only cellular mechanism regulated by small pieces of RNA. Thanks to groundbreaking work in C. elegans , researchers have discovered a class of natural small RNA molecules called microRNAs that appear to be crucial in regulating development. Although they apparently use the same tools as siRNAs to carry out their functions—Dicer, RISC, and a family of proteins known as Argonautes—microRNAs differ in that their sequences do not precisely match their mRNA targets. As such, they regulate the expression of proteins by those targets, rather than degrade them altogether, which leads investigators to believe that they play an important role in the timing and nature of development—perhaps to the point of controlling differentiation in embryonic stem cells.
    • RNA interference and human disease. Cheng JC, Moore TB, Sakamoto KM. Mattel Children's Hospital, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA).The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine. PMID: 14567961 [PubMed - in process]
    • Targeted gene silencing by small interfering RNA-based knock-down technology. Zhang J, Hua ZC. State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing, 210093 China. RNA interference (RNAi) has emerged as a powerful tool for the silencing of gene expression in animals and plants. RNAi is mediated by approximately 21-nt small interfering RNAs (siRNAs), which are originally produced from larger double stranded RNAs (dsRNAs) in vivo through the action of Dicer. Recently, many groups have reported systems designed to express siRNAs in mammalian cells through transfection of either oligonucleotides or plasmids encoding siRNAs. Although the use of siRNAs to silence genes in vertebrate cells was only reported three years ago, the emerging literature indicates that most vertebrate genes can be studied with this technology. This review summarizes some approaches to generate siRNAs, the delivery and application of siRNAs to target cells and the utility of siRNAs as analytical and potential therapeutic tools. PMID: 14965205 [PubMed - in process]
      • Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Myers JW, Jones JT, Meyer T, Ferrell JE Jr. Department of Molecular Pharmacology, 269 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5174, USA. jmyers@stanford.edu RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs. Publication Types:
      • Evaluation Studies
      • Technical Report
      • PMID: 12592410 [PubMed - indexed for MEDLINE]
      • Retroviral delivery of small interfering RNA into primary cells. Barton GM, Medzhitov R. Section of Immunobiology and The Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520, USA. RNA interference is an evolutionarily conserved process in which recognition of double-stranded RNA ultimately leads to posttranscriptional suppression of gene expression. This suppression is mediated by short (21- to 22-nt) small interfering RNAs (siRNAs), which induce degradation of mRNA based on complementary base pairing. The silencing of gene expression by siRNAs is emerging rapidly as a powerful method for genetic analysis. Recently, several groups have reported systems designed to express siRNAs in mammalian cells through transfection of either oligonucleotides or plasmids encoding siRNAs. Because these systems rely on transfection for delivery, the cell types available for study are restricted generally to transformed cell lines. Here, we describe a retroviral system for delivery of siRNA into cells. The use of retroviral vectors can greatly expand the types of cells available for RNA interference analysis. Furthermore, we demonstrate that this retroviral system allows for stable inactivation of genes in primary cells. PMID: 12417750 [PubMed - indexed for MEDLINE]
      • Gene silencing mediated by small interfering RNAs in mammalian cells. Scherr M, Morgan MA, Eder M. Hannover Medical School, Department of Hematology and Oncology, 30625 Hannover, Germany. M.Scherr@t-online.de RNA interference (RNAi) as a protecting mechanism against invasion by foreign genes was first described in C. elegans and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. In mammalian cells siRNA molecules are capable of specifically silencing gene expression without induction of the unspecific interferon response pathway. Thus, siRNAs have become a new and powerful alternative to other genetic tools such as antisense oligonucleotides and ribozymes to analyze loss-of-function phenotypes. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into target cells and for some applications long-term siRNA expression. Effective strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. This review summarizes some genetic and biochemical aspects of RNAi, the delivery and application of siRNAs to target cells, the kinetics of RNAi and the utility of siRNAs as analytical and potential therapeutic tools.
      • Publication Types:
      • Review
      • Review, Tutorial
      • PMID: 12570711 [PubMed - indexed for MEDLINE]
      • RNA interference (RNAi) in hematology. Scherr M, Steinmann D, Eder M. Department of Hematology and Oncology, Hannover Medical School, Carl-Neuberg Strasse1, 30625 Hannover, Germany. M.Scherr@t-online.de RNA interference (RNAi), an evolutionary highly conserved process of post-transcriptional gene silencing, can be triggered by small interfering RNAs (siRNAs) that mediate sequence-specific mRNA degradation. Since the first reports in 1998, RNAi has rapidly been developed into an effective tool to specifically knock down gene expression in a wide variety of target cells. Accordingly, RNAi is currently used for both systematic functional genomics in several organisms and for specific therapeutic intervention in preclinical models of different diseases characterized by aberrant gene expression. However, since siRNAs are not replicated in mammalian cells during the process of RNAi, kinetic aspects of RNAi-induced gene silencing that eventually depend on the intracellular level of siRNA must be considered for each analytical or therapeutic application in these cells. We summarize here some aspects of siRNA design and delivery, of RNAi kinetics, potential side effects, and limitations of siRNA-mediated gene silencing, as well as putative RNAi targets for functional and/or therapeutic intervention in hematopoietic cells. Publication Types:
      • Review
      • Review Literature
      • PMID: 14574462 [PubMed - indexed for MEDLINE]
    • siRNA function in RNAi: a chemical modification analysis. Chiu YL, Rana TM. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications. PMID: 12923253 [PubMed - indexed for MEDLINE]
    • Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs. Hamada M, Ohtsuka T, Kawaida R, Koizumi M, Morita K, Furukawa H, Imanishi T, Miyagishi M, Taira K. Biomedical Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan. The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3'-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3'-end of either strand, using 2'-O,4'-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3'-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3'-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3'-end is recognized by the RNA-induced silencing complex (RISC). PMID: 12477280 [PubMed - indexed for MEDLINE]
      • Gene silencing in mammals by small interfering RNAs. McManus MT, Sharp PA. Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames Street E17-526, Cambridge, Massachusetts 02139, USA. mmcmanus@mit.edu Among the 3 billion base pairs of the human genome, there are approximately 30,000-40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology. Publication Types:
      • Review
      • Review, Tutorial
      • PMID: 12360232 [PubMed - indexed for MEDLINE]
    • RNA interference by short hairpin RNAs expressed in vertebrate cells. Hannon GJ, Conklin DS. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA. RNA interference (RNAi) is now established as a general method to silence gene expression in a variety of organisms. Double-stranded RNA (dsRNA), when introduced to cells, interferes with the expression of homologous genes, disrupting their normal function. In mammals, transient delivery of synthetic short interfering RNAs (siRNAs), which resemble the processed form of standard double stranded RNAi triggers, is effective in silencing mammalian genes. Issues related to transfer efficiency and duration of the silencing effect, however, restrict the spectrum of the applications of siRNAs in mammals. These shortcomings of siRNAs have been solved by the cellular expression of short hairpin RNAs (shRNAs) from DNA vectors. shRNAs are indistinguishable from siRNAs in terms of efficacy and mechanism but can be produced within cells from standard mammalian expression vectors. In this way, shRNA expression makes possible the creation of continuous cell lines and transgenic animals in which suppression of a target gene is stably maintained by RNAi. As a result, the types of RNAi-based gene function analysis that can be carried out in mammals have been greatly expanded. We describe methods for the construction and transfer of stable shRNA expressing vectors suitable for generating loss of function alleles in mammalian cells in vitro or in vivo. PMID: 14770011 [PubMed - in process]
    • siRNA Target Finding Strategy (1): Thermodynamic Property - Sequences were filtered to remove candidates with unfavored thermodynamic property (2,4,5) Length of the siRNA target site: - Default is 21 mer (3). GC content of the target site - Default GC% range is 30-60%. Sequences with low GC content (<60%) have a better chance to be functional siRNAs site than those with high GC content (>60%). For GC rich genes, the upper limit of the GC% range may be adjusted to a higher level. Sequence region - We recomment ORF (open reading frame) as the target selecting region, preferably 50-100 nt downstream of the &quot;ATG&quot; start codon, and you may specify your desired region in our design tool. Organism - The human, mouse, and rat genomes are currently available. The specified genome is used to remove non-unique target sequences via BLAST search. RNA secondary Structure - We calculated the RNA secondary structure and minimum free energy (MFE) for each target sense and anti-sense sequences. Sequences with relatively strong internal structures will be removed. Repeat and Low Complex Sequence - Sequences with tandem repeats and other low complex features will be removed. SNP - Sequences overlapping with a SNP site will be marked and filtered. This is done only when an accession/gene id is provided. BLAST search - Each candidate target sequence will be searched against a unique and comprehensive EST/mRNA collection for the specified organism. Sequences overlapping 17 bp or more with other transcripts are removed from the final list. ΔE and Ranking - The candidates are ranked based on a GenScript proprietary algorithm and using the paramter ΔE. This paramter measures the specificity of a target site. References : 1. Wang L, Mu FY. (2004) A Web-based Design Center for Vector-based siRNA and siRNA cassette. Bioinformatics. (In press) 2. Khvorova A, Reynolds A, Jayasena SD. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell. 115(2):209-16. 3. Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 13(2):83-105. 4. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. (2004) Rational siRNA design for RNA interference. Nat Biotechnol. 22(3):326-30. 5. Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32(3):936-48.
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    • RIBONUCLEIC ACID STRUCTURE OF THE SPECIFICITY DOMAIN OF RIBONUCLEASE P RNA PDB code: INXL Authors: A. S. Krasilnikov, X. Yang, T. Pan, A. Mondragon Date: 10 Feb 03 Method: THEORETICAL MODEL Resolution: Not Applicable. Source: 1. Bacillus subtilis; Bacteria; Bacillus Subtilis; Bacteria; Puc19; The RNA Was Produced By Vitro Transcription With T7 Polymerase From A Puc19-Based Template Which Contained Cloned Sequence Of The S-Domain. Keywords: Ribonuclease P RNA, P RNA, S-Domain (Keyword Search: [Gene Ontology/PubMed/Web (Google)]) ENGINEERED: Yes; CHAIN: M; MOLECULE: Ribonuclease P RNA; MOL ID: Mol_id: 1; MUTATION: Yes FRAGMENT: Specificity Domain, S-Domain; Ligands, modified residues, ions: MG: Magnesium Ion Sequence, Chains, Units Reference: A. S. Krasilnikov, X. Yang, T. Pan, A. Mondragon Crystal Structure Of The Specificity Domain Of Ribonuclease P Nature V. 421 760 2003 (for further references see the PDB file header)
      • Nature Reviews Molecular Cell Biology 4 , 457 -467 (2003); doi:10.1038/nrm1129  
      • KILLING THE MESSENGER: SHORT RNAS THAT SILENCE GENE EXPRESSION
      • Derek M. Dykxhoorn, Carl D. Novina & Phillip A. Sharp    
      • Preface
      • Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.
      • Summary
        • 21–23-nucleotide (nt) double-stranded (ds), short interfering (si)RNAs can silence gene expression by directing the sequence-specific cleavage of target messenger RNA in a process that is referred to as RNA interference (RNAi).
        • Although long dsRNAs have been used successfully for the silencing of gene expression in various organisms including Caenorhabditis elegans , plants, Drosophila and mouse oocytes, their use in mammalian systems has been limited primarily because the introduction of dsRNA longer than 30 nt induces a nonspecific interferon response.
        • The evidence that siRNAs could be introduced into mammalian cells and cause the sequence-specific degradation of mRNA without inducing an interferon response has revolutionized the way in which mammalian somatic-cell genetic studies are approached, laid the ground work for the development of short-RNA-based silencing technologies, and holds promise for the development of potential siRNA-based therapeutic strategies.
        • The use of synthetic siRNAs for gene silencing leads to a transient response. DNA-vector-based delivery systems have been developed, which prolonged the silencing effect indefinitely.
        • Most of these expression systems take advantage of the ability of Dicer to process short hairpin RNAs into siRNAs and thereby silence the target gene. These technologies have made it possible to carry out gene-silencing experiments in various cell types and cell lines, as well as to create transgenic animals that stably silence a target gene.
        • This article discusses the rapid advances in RNAi-based gene-silencing technologies and the impact these advances have on the study of gene function, including the development of transgenic animals, siRNA-mediated gene silencing in somatic tissues and functional-genomic studies.
    • BIOTECHNOLOGY VIRUS ALERT Louisa Flintoft Nature Publishing Group RNA interference (RNAi) — the process in which double-stranded RNA (dsRNA) binds to specific messenger RNA and silences gene expression — has generated great excitement, as dsRNA could theoretically be created to target any disease-causing gene more quickly, cheaply and effectively than any present method. Preliminary in vivo work seems to be maintaining this promise. Earlier this year it was shown that RNAi can silence the expression of the gene encoding the Fas receptor, protecting mice from fulminant hepatitis. Now, Kay and colleagues report for the first time in Nature Biotechnology that RNAi can prevent viral replication in live mammals.   The authors used a mouse model of hepatitis B virus (HBV) infection, in which a plasmid carrying the entire HBV genome was transfected into the mouse liver. In these mice, HBV proteins are expressed and most of the stages of viral replication take place. Kay and colleagues co-transfected the mice with a second plasmid that expressed small fragments of dsRNA called short hairpin RNAs (shRNAs), designed to target specific HBV mRNA sequences. This caused a dramatic decrease in viral gene expression at the levels of RNA and protein, and strongly inhibited the replication of the virus. One of the concerns about testing RNAi in whole animals is that any observed effect could result, fully or in part, from the foreign shRNAs triggering the immune response. But by comparing mice that lack a functional immune system with wild-type animals, the authors showed that the HBV inhibition was a specific effect of targeting viral sequences, rather than being due to a generalized immune-based attack. At present, there are few effective treatments that can treat hepatitis B once infection is established. The findings not only show the therapeutic promise of RNAi in hepatitis B but also indicate that this strategy could treat other forms of viral infection. But many challenges remain. First, RNAi-based inhibition of infection by intact HBV needs to be proved. Second, new methods of delivery will need to be developed, as the one used in this study is unsuitable for a clinical setting. And finally, successfully silencing the HBV genes of interest without affecting the expression of any host genes must be ensured. Nature Reviews Drug Discovery 2 , 512 (2003); doi:10.1038/nrd1143
    • TECHNIQUE DOWN A HAIRPIN Arianne Heinrichs Short hairpin RNAs (shRNAs) — synthetic molecules that are modelled on small, non-coding microRNA molecules with a 'hairpin' secondary structure — can silence gene expression by RNA interference (RNAi), much as small interfering RNAs (siRNAs) do. As now reported in Nature Structural Biology , Thomas Rosenquist's group, in collaboration with Greg Hannon's group, explored whether germline transmission of shRNA constructs was feasible in mammals, as this would enable stable, long-term silencing of gene expression.   The initial attempt of Rosenquist and colleagues to achieve germline transmission using standard transgenics methods, in which linearized constructs were injected into pronuclei to create transgenic founder animals, was unsuccessful. Choosing Neil1 (a DNA N -glycosylase that initiates base-excision repair) as the target gene, they turned to a different approach — based on the use of embryonic stem (ES) cells. The authors created a single shRNA expression construct against the Neil1 target gene, which was introduced into mouse ES cells by electroporation. Stable ES cell lines showed 80% reduction of Neil1 protein, which correlated with a similar reduction in mRNA levels, and the cells were approximately twofold more sensitive to ionizing radiation — consistent with Neil1's role in DNA repair. To obtain transgenic animals, the authors injected cells from two independent ES cell lines into blastocysts. The chimeric mice that contained a high percentage of ES-derived cells were outcrossed, and germline transmission of the shRNA expression construct was detected in several F1 offspring. Neil1 protein and mRNA levels were reduced in several tissues of F1 mice, as was observed in the ES cells. In addition, siRNA was detected, by northern blotting, in animals that carried the shRNA expression vector, but not in those that lacked the vector. The authors conclude, therefore, that shRNAs can be used to create germline transgenic mice in which a target gene is silenced by RNAi. These findings now open the door for tissue-specific, inducible and reversible suppression of gene expression in mice. Nature Reviews Drug Discovery 2 , 530 (2003); doi:10.1038/nrd1143
      • DISSECTING HIV-1 THROUGH RNA INTERFERENCE Mario Stevenson   
      • Preface
      • In cells of organisms, ranging from nematodes to primates, there is a process known as RNA interference (RNAi) that effects the degradation of RNA in a highly sequence-specific manner. Scientists have figured out a way to co-opt elements of the RNAi machinery such that almost any RNA can be targeted for degradation. It is now clear that HIV-1 is fair game for RNAi; viral RNA intermediates have been targeted as well as messenger RNAs for cellular co-factors that are required for replication of HIV-1. The hope is that RNAi can be used not only as a research tool, but also as a therapeutic strategy for infection with HIV-1.
      • Summary
        • RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) induces silencing of homologous, endogenous and exogenous genes. This offers a powerful process to regulate gene expression to determine the role of specific proteins in biological processes.
        • The replication of viruses, such as HIV-1, depends on cellular factors that can oppose or enhance virus replication. RNAi can be used to manipulate the expression of these cellular factors to examine their role in virus replication.
        • Several strategies are being developed to introduce interfering RNAs into cells that are normally infected by viruses, so facilitating the study of HIV biology by RNAi.
        • The use of RNAi as a therapeutic approach to virus infections such as AIDS faces several obstacles. However, because of its mechanism of action, RNAi-based therapeutic approaches might prove effective where other antiviral agents in use at present have failed.
      Nature Reviews Immunology 3 , 851 -858 (2003); doi:10.1038/nri1227
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