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Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

Abd Ellah N, Taylor L, Troja W, Owens K, Ayres N, Pauletti G, Jones H - PLoS ONE (2015)

Bottom Line: Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease.To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta.IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth.

View Article: PubMed Central - PubMed

Affiliation: James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, 45267, United States of America; Faculty of Pharmacy, Assiut University, 71515, Assiut, Arab Republic of Egypt.

ABSTRACT
Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

No MeSH data available.


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(A) The HPMA-DMAEMA copolymer used for DNA delivery in both in vitro and in vivo studies. (B) Maps of the CMV-eGFP and Trophoblast-specific plasmids.
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pone.0140879.g001: (A) The HPMA-DMAEMA copolymer used for DNA delivery in both in vitro and in vivo studies. (B) Maps of the CMV-eGFP and Trophoblast-specific plasmids.

Mentions: All starting materials were purchased from Aldrich at the highest purity available and used as received unless specified otherwise. Synthesis of S-1-ethyl-S’-(α’,α’-dimethyl-α”-acetic acid)trithiocarbonate (EDMAT) was adapted from the procedure described by Convertine et al. [19]. Synthesis of 2-Hydroxypropyl methacrylamide (HPMA) monomer was adapted from the procedure described by Rowe et al. [20]. Polymerization of HPMA with RAFT agent was adapted from the procedure described by Rowe et al. [20], briefly, the EDMAT RAFT agent (0.0314 g, 0.001 mol), HPMA (4.0 g, 0.03 mol), AIBN (0.0023 g, 0.0001 mol), and tert-butanol (27 mL, 0.28 mol) were added to a 100 mL round-bottom flask equipped with a stir bar. The reaction was placed in an ice bath and the reaction mixture was purged under nitrogen for 45 min and left under an N2 atmosphere. The reaction was transferred to an 80°C oil bath and allowed to react for 6 h, sampling every hour. The reaction was quenched by exposure to air and precipitated from ether to yield a pale yellow polymer. Isolated yield: 40%. PDI = 1.24. Mn = 13000 g/mol. 1H NMR (CDCl3): 1.1 (d, 76H), 1.3 (s, 3H), 3.19 (t, 1H), 3.95 (d, 2H), 6.58 (br, 1H). FT-IR (cm-1): ν(OH) = 3323.3 broad, ν(CH = CH2) = 2979.9 and 2291.1, ν (NHC = O) = 1672.4. Polymerization of DMAEMA with HPMA macro-RAFT agent was adapted from the procedure described by Duvall et al. [21]. The HPMA macro-RAFT agent (0.07 mmol), 2-(dimethylamino)ethyl methacrylate (DMAEMA) (0.012 mol), 2,2’-azobis(2-methylpropionitrile) (AIBN) (0.077 mmol), and dimethylformamide (0.15 mol) were added to a 100mL round-bottom flask equipped with a stir bar. The reaction was placed in an ice bath and purged under nitrogen for 45 min and left under an N2 atmosphere. The reaction was transferred to a 65°C oil bath and allowed to polymerize for 6 h. The reaction was quenched by exposure to air and precipitated from ethyl ether:pentane (50:50). The resulting copolymer structure is shown in Fig 1.


Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

Abd Ellah N, Taylor L, Troja W, Owens K, Ayres N, Pauletti G, Jones H - PLoS ONE (2015)

(A) The HPMA-DMAEMA copolymer used for DNA delivery in both in vitro and in vivo studies. (B) Maps of the CMV-eGFP and Trophoblast-specific plasmids.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608830&req=5

pone.0140879.g001: (A) The HPMA-DMAEMA copolymer used for DNA delivery in both in vitro and in vivo studies. (B) Maps of the CMV-eGFP and Trophoblast-specific plasmids.
Mentions: All starting materials were purchased from Aldrich at the highest purity available and used as received unless specified otherwise. Synthesis of S-1-ethyl-S’-(α’,α’-dimethyl-α”-acetic acid)trithiocarbonate (EDMAT) was adapted from the procedure described by Convertine et al. [19]. Synthesis of 2-Hydroxypropyl methacrylamide (HPMA) monomer was adapted from the procedure described by Rowe et al. [20]. Polymerization of HPMA with RAFT agent was adapted from the procedure described by Rowe et al. [20], briefly, the EDMAT RAFT agent (0.0314 g, 0.001 mol), HPMA (4.0 g, 0.03 mol), AIBN (0.0023 g, 0.0001 mol), and tert-butanol (27 mL, 0.28 mol) were added to a 100 mL round-bottom flask equipped with a stir bar. The reaction was placed in an ice bath and the reaction mixture was purged under nitrogen for 45 min and left under an N2 atmosphere. The reaction was transferred to an 80°C oil bath and allowed to react for 6 h, sampling every hour. The reaction was quenched by exposure to air and precipitated from ether to yield a pale yellow polymer. Isolated yield: 40%. PDI = 1.24. Mn = 13000 g/mol. 1H NMR (CDCl3): 1.1 (d, 76H), 1.3 (s, 3H), 3.19 (t, 1H), 3.95 (d, 2H), 6.58 (br, 1H). FT-IR (cm-1): ν(OH) = 3323.3 broad, ν(CH = CH2) = 2979.9 and 2291.1, ν (NHC = O) = 1672.4. Polymerization of DMAEMA with HPMA macro-RAFT agent was adapted from the procedure described by Duvall et al. [21]. The HPMA macro-RAFT agent (0.07 mmol), 2-(dimethylamino)ethyl methacrylate (DMAEMA) (0.012 mol), 2,2’-azobis(2-methylpropionitrile) (AIBN) (0.077 mmol), and dimethylformamide (0.15 mol) were added to a 100mL round-bottom flask equipped with a stir bar. The reaction was placed in an ice bath and purged under nitrogen for 45 min and left under an N2 atmosphere. The reaction was transferred to a 65°C oil bath and allowed to polymerize for 6 h. The reaction was quenched by exposure to air and precipitated from ethyl ether:pentane (50:50). The resulting copolymer structure is shown in Fig 1.

Bottom Line: Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease.To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta.IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth.

View Article: PubMed Central - PubMed

Affiliation: James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, 45267, United States of America; Faculty of Pharmacy, Assiut University, 71515, Assiut, Arab Republic of Egypt.

ABSTRACT
Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

No MeSH data available.


Related in: MedlinePlus