Viral Gene Therapy to Brain Capillary Endothelial Cells for Protein Secretion of Recombinant Niemann-Pick Type C2 Protein - A Novel Strategy for Delivery of NPC2 to the Brain
Author
Olsen, Eva Hede
Term
4. term
Publication year
2017
Submitted on
2017-12-02
Pages
65
Abstract
Det er vanskeligt at få lægemidler ind i hjernen, fordi blod-hjerne-barrieren (BBB) blokerer for de fleste stoffer. En ny strategi er at ændre hjernens blodkars endotelceller (BCECs) med en virusbaseret metode, så de selv kan udskille terapeutiske, rekombinante proteiner til centralnervesystemet. En relevant anvendelse er Niemann-Pick type C2, en arvelig sygdom med mangel på det opløselige NPC2-protein, som fører til kolesterolophobning i cellers lysosomer, viscerale symptomer og progressiv neurodegeneration. Lysosomal lagring er forbundet med øget dannelse af lysosomer og højere udtryk af lysosomale gener. Intravenøs erstatning af NPC2 kan forbedre viscerale, men ikke CNS-symptomer, fordi proteinet ikke passerer BBB. Formålet var derfor at undersøge, om BCECs kan genetisk modificeres med en lentiviral vektor til at udskille rekombinant NPC2 mod både hjernesiden og kredsløbet uden at svække barrieren i en in vitro BBB-model, og om det udskilte NPC2 kan reducere kolesterolophobning i NPC2-muterede humane fibroblaster. NPC2-udtrykkende lentivirale vektorer blev produceret og brugt til at transducere en in vitro BBB-model. Effektiviteten blev vurderet med flowcytometri og immunocytokemisk farvning for NPC2. Barriereintegritet blev løbende målt med transendothelial elektrisk modstand (TEER). Konditioneret medium fra transducerede celler blev brugt til at behandle NPC2-muterede fibroblaster, og kolesterol blev vurderet med Filipin III-farvning. Udtrykket af de lysosomale gener Lamp1 og MCOLN1 blev desuden målt i wildtype- og NPC2-muterede fibroblaster med og uden NPC2-tilførsel. BBB-modellen blev vellykket transduceret uden tab af barriereintegritet. Den lentivirale vektor gav mere stabil, langvarig NPC2-ekspression end en ikke-viral vektor. Konditioneret medium fra genetisk modificerede celler reducerede kolesterolophobning i NPC2-muterede fibroblaster, men en lignende effekt sås med medium fra ikke-modificerede celler på grund af uventet høj naturlig sekretion af endogent NPC2. Der sås dog tegn på terapeutisk effekt af udskilt rekombinant NPC2 ved behandling med konditioneret medium fra det øverste kammer i den genetisk modificerede BBB-model. MCOLN1-udtrykket var øget i NPC2-muterede fibroblaster sammenlignet med wildtype og syntes at falde efter NPC2-tilførsel, mens der ikke sås signifikante forskelle for Lamp1.
Delivering medicines to the brain is challenging because the blood–brain barrier (BBB) blocks most compounds. A new approach is to use virus-based gene delivery to brain endothelial cells (BCECs) so they secrete therapeutic, recombinant proteins into the central nervous system. A relevant application is Niemann–Pick type C2, a genetic disease caused by deficiency of the soluble NPC2 protein, which leads to cholesterol accumulation in lysosomes, visceral symptoms, and progressive neurodegeneration. Lysosomal storage is associated with increased lysosome biogenesis and higher expression of lysosomal genes. Intravenous NPC2 replacement can reverse visceral but not CNS symptoms because NPC2 does not cross the BBB. This study therefore examined whether BCECs can be genetically modified with a lentiviral vector to secrete recombinant NPC2 toward both the brain side and the circulation without weakening the barrier in an in vitro BBB model, and whether secreted NPC2 can reduce cholesterol accumulation in NPC2 mutant human fibroblasts. NPC2-expressing lentiviral vectors were produced and used to transduce an in vitro BBB model. Gene modification efficiency was evaluated by flow cytometry and immunocytochemical staining for NPC2. Barrier integrity was monitored by transendothelial electrical resistance (TEER). Conditioned medium from transduced cells was used to treat NPC2 mutant fibroblasts, and cholesterol was assessed by Filipin III staining. Relative expression of the lysosomal genes Lamp1 and MCOLN1 was also measured in wild-type and NPC2 mutant fibroblasts with and without NPC2 replacement. The BBB model was successfully transduced without compromising barrier integrity. Lentiviral delivery supported more stable, long-term NPC2 expression than a non-viral approach. Conditioned medium from genetically modified cells reduced cholesterol accumulation in NPC2 mutant fibroblasts, but a similar effect was seen with medium from non-modified cells due to unexpectedly high natural secretion of endogenous NPC2. Nevertheless, conditioned medium collected from the top chamber of the genetically modified BBB model showed signs of a therapeutic effect from secreted recombinant NPC2. MCOLN1 expression was higher in NPC2 mutant fibroblasts than in wild type and appeared to decrease with NPC2 replacement, while no significant differences were observed for Lamp1.
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