Levering af terapeutisk siRNA til CNS Brug kationiske og anioniske liposomer
Summary
The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.
Abstract
Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects.
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.
Introduction
Prioner er alvorlige neurodegenerative sygdomme, der påvirker CNS. Prionsygdomme følge af fejlfoldning af den normale cellulære prionprotein, PrP C, af et infektiøst isomer kaldet PrP Res. Disse sygdomme rammer en bred vifte af arter, herunder bovin spongiform encephalopati i køer, scrapie, chronic wasting disease hos hjortedyr, og Creutzfeldt-Jakobs sygdom hos mennesker 1-3. Prioner forårsage neurodegeneration, der starter med synaptisk tab, og udvikler sig til vakuolisering, gliose, neuronal tab, og plaque indskud. Til sidst, hvilket resulterer i død af dyret / individuelle 4. I årtier har forskere undersøgt forbindelser beregnet til at bremse eller stoppe progressionen af prion sygdom. Men forskere har ikke fundet enten en vellykket terapi eller en effektiv systemisk levering køretøj.
Endogen PrP C-ekspression er nødvendig for udviklingen af prionsygdomme 5 </sop>. Derfor kan faldende eller eliminere PrP ° ekspression medføre en forsinkelse eller lindring af sygdom. Flere grupper skabt transgene mus med reducerede niveauer af PrP ° eller injicerede lentivectors udtrykker shRNA direkte i murine hjernevæv for at undersøge den rolle, som PrP C ekspressionsniveauer i prionsygdom. Disse forskere fundet at reducere mængden af neuronal PrP C resulterede i at standse den gradvise neuropatologi af prionsygdomme og forlænget dyrenes liv 6-9. Vi har rapporteret, at PrP C siRNA behandlingsresultater i clearance af PrP Res i mus neuroblastomceller 10. Disse undersøgelser antyder, at anvendelse af terapier for at formindske PrP C ekspressionsniveauer, ligesom små interfererende RNA (siRNA), som spalter mRNA, kan tilstrækkeligt forsinke progressionen af prionsygdomme. Imidlertid blev de fleste terapier undersøgt for prionsygdomme leveret på måder, som ikke ville være praktiski et klinisk miljø. Derfor er en siRNA terapi behov for et systemisk leveringssystem, som leveres intravenøst og målrettet til CNS.
Forskere har undersøgt anvendelsen af liposomer som leveringsvehikler til genterapi produkter. Kationiske og anioniske lipider begge anvendes i dannelsen af liposomer. Kationiske lipider er mere udbredt end anioniske lipider fordi ladningen forskellen mellem det kationiske lipid og DNA / RNA muliggør effektiv emballage. En anden fordel ved kationiske lipider er, at de krydser cellemembranen lettere end andre lipider 11-14. Men kationiske lipider er mere immunogene end anioniske lipider 13,14. Derfor har forskere begyndt at skifte fra at bruge kationiske til anioniske lipider i liposomer. Genterapiprodukter effektivt kan pakkes i anioniske liposomer under anvendelse af positivt ladede peptid protaminsulfat, som kondenserer DNA / RNA-molekyler 15-19. Da anioniske Lipids er mindre immunogene end kationiske lipider, de kan have øget cirkulation gange, og kan være mere tolereres i dyremodeller 13,14. Liposomer er målrettet til specifikke væv ved hjælp af målrettet peptider, der er knyttet til liposomerne. RVG-9r neuropeptid, som binder til nicotinacetylcholinreceptorer, er blevet anvendt til at målrette siRNA og liposomer til CNS 17-20.
Denne rapport skitserer en protokol til at producere tre siRNA levering køretøjer, og til at pakke og levere siRNA til neuronale celler (Figur 1). Liposom-siRNA-peptidkomplekser (LSPCs) er sammensat af liposomer med siRNA og RVG-9r målrettende peptid elektrostatisk bundet til den ydre overflade af liposomet. Peptid rettet liposomindkapslet terapeutiske siRNA (PALETS) er sammensat af siRNA og protamin indkapslet i liposomet, med RVG-9r kovalent bundet til lipid PEG-grupper. Brug nedenstående metoder til at generere LSPCs og PALETS, PrP ° siRNA falder PrP ° ekspression op til 90% i neuronale celler, der besidder et fantastisk løfte at helbrede eller i det væsentlige sinke udviklingen af prionsygdom patologi.
Protocol
Representative Results
Discussion
Denne rapport beskriver en protokol til at oprette to målrettede doseringssystemer, der effektivt transporterer siRNA til CNS. Tidligere metoder til at levere siRNA til CNS inkluderet injicere siRNA / shRNA vektorer direkte ind i hjernen, intravenøs injektion af målrettet siRNA eller intravenøs injektion af ikke-målrettede liposom-siRNA komplekser. Injektion af siRNA / shRNA vektorer i CNS forårsager et fald i målprotein ekspressionsniveauer. Men den siRNA / shRNA ikke diffundere frit gennem CNS. Desuden er disse…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.
Materials
| DOTAP lipid | Avanti Lipids | 890890 | |
| Cholesterol | Avanti Lipids | 700000 | |
| DSPE | Avanti Lipids | 850715 | |
| DSPE-PEG | Avanti Lipids | 880125 | |
| Chloroform | Fisher Scientific | AC268320010 | |
| Methanol | EMD Millipore | 113351 | |
| N2 Gas | AirGas | ||
| Sucrose | Fisher Scientific | S5-500 | |
| Extruder | Avanti Lipids | 610023 | |
| 1.0, 0.4, and 0.2um filters | Avanti Lipids | 610010, 610007, 610006 | |
| PBS | Life Technologies | 70011-044 | |
| Protamine sulfate | Fisher Scientific | ICN10275205 | |
| EDC | Thermo Scientific | 22980 | Aliquoted for single use |
| Sulfo-NHS | Thermo Scientific | 24510 | Aliquoted for single use |
| 40um Cell Strainer | Fisher Scientific | 08-771-1 | |
| Rat anti-mouse CD16/CD32 Fc block | BD Pharmigen | 553141 | |
| Anti-PrP antibody (PRC5) | Proprietary – PRC |
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