KR20240051530A - Pharmaceutical composition comprising PrPc protein using recombinant adenovirus vector for preventing, diagnosis or treating Japanese encephalitis virus infection disease - Google Patents
Pharmaceutical composition comprising PrPc protein using recombinant adenovirus vector for preventing, diagnosis or treating Japanese encephalitis virus infection disease Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
본 발명은 재조합 아데노바이러스 벡터를 이용한 PrPc 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방, 진단 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection containing PrPc protein as an active ingredient using a recombinant adenovirus vector.
Description
본 발명은 재조합 아데노바이러스 벡터를 이용한 PrPc 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방, 진단 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection containing PrPc protein as an active ingredient using a recombinant adenovirus vector.
일본뇌염바이러스(JEV)는 ZIKA 바이러스(ZIKV), 웨스트 나일 바이러스(WNV), 뎅기열 바이러스(DENV) 등을 포함하는 플라비바이러스 아과(Flaviviridae)에 의해 유발되는 급성 모기 매개 바이러스성 질병이다. 일본 뇌염 바이러스는 뇌 감염을 유발한다. 일본 뇌염 바이러스는 아시아 태평양 및 호주의 많은 지역에서 풍토병으로 간주된다. 현재 일본 뇌염은 전체 환자의 약 25%가 사망하고, 약 25%는 낫더라도 지적 장애나 손발 마비 등의 후유증을 남기며, 나머지 약 50%만 완쾌되는 질병이다. 마비, 중추신경계 이상, 기면증, 섬망 등이 나타나고, 세균 감염에 의해 호흡 곤란을 동반한 폐렴 증상이 나타나기도 한다. 백신 접종으로 감염을 사전에 예방하지만 현재 일본 뇌염만을 치료하는 적절한 치료제는 없다. 플라비 바이러스에 대한 대책으로는 효능이 높은 백신을 개발, 사용하는 것이 우선적이지만, 이와 함께 이들 바이러스에 대해서 광범위하게 작용하는 치료 물질의 개발이 필요하다. Japanese encephalitis virus (JEV) is an acute mosquito-borne viral disease caused by the Flaviviridae subfamily, which includes ZIKA virus (ZIKV), West Nile virus (WNV), and dengue virus (DENV). Japanese encephalitis virus causes brain infection. Japanese encephalitis virus is considered endemic in many parts of Asia Pacific and Australia. Currently, Japanese encephalitis is a disease in which about 25% of all patients die, and even if about 25% recover, it leaves aftereffects such as intellectual disability or paralysis of the hands and feet, and only about 50% of the remaining patients recover completely. Paralysis, central nervous system abnormalities, narcolepsy, and delirium may occur, and symptoms of pneumonia accompanied by breathing difficulties may also appear due to bacterial infection. Infection is prevented through vaccination, but there is currently no appropriate treatment for Japanese encephalitis. As a countermeasure against flaviviruses, the development and use of highly effective vaccines is a priority, but it is also necessary to develop therapeutic substances that act broadly against these viruses.
정상적인 프리온 단백질 (PrPC)은 뉴런에 의해 가장 풍부하게 발현되며, 비정상적으로 접힌 아밀로이드 생성 동형체로서의 구조적 전환은 인간의 크로이츠펠트-야콥병(CJD) 및 쿠루, 소 해면상 뇌병증(BSE)를 포함하는 신경퇴행성 장애 그룹인 프리온 질병의 발병에서 중요한 병원성 사건이다. PrPC가 수십 년 동안 광범위하게 연구되었지만 일반적인 기능은 아직 완전히 이해되지 않았다. Normal prion protein (PrPC) is most abundantly expressed by neurons, and its structural transition to an abnormally folded amyloidogenic isoform is responsible for neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD) in humans and kuru, bovine spongiform encephalopathy (BSE). It is an important pathogenic event in the pathogenesis of the disorder group prion diseases. Although PrP C has been studied extensively for decades, its general functions are not yet fully understood.
본 발명자들은 뉴런 세포에서 JEV의 감염에 의한 치료제를 연구한 결과, JEV에 의한 프리온 단백질 분해는 뉴런에서 오토파지의 활성화를 통해 세포 사멸을 유도하는 것을 확인하고, 재조합 아데노바이러스 벡터를 이용하여 Prnp를 형질 감염하여 PrPC 의 단백질 수준이 증가된 세포에 JEV를 감염시킨 결과, 세포내 오토파지가 억제되고, 일본 뇌염 바이러스의 RNA와 단백질의 수준이 감소하는 것을 확인함으로써 본 발명을 완성하였다. As a result of studying a treatment for JEV infection in neuronal cells, the present inventors confirmed that prion protein degradation by JEV induces cell death through activation of autophagy in neurons, and used a recombinant adenovirus vector to transform Prnp. The present invention was completed by infecting cells with increased PrP C protein levels by transfection with JEV and confirming that intracellular autophagy was inhibited and the levels of RNA and protein of Japanese encephalitis virus were decreased.
본 발명은 재조합 아데노바이러스 벡터를 이용한 PrPc 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방, 진단 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다. The purpose of the present invention is to provide a pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection containing PrPc protein as an active ingredient using a recombinant adenovirus vector.
본 발명은 재조합 아데노바이러스 벡터를 이용한 PrPc 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방, 진단 또는 치료용 약학적 조성물을 제공할 수 있다. The present invention can provide a pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection containing PrPc protein as an active ingredient using a recombinant adenovirus vector.
본 발명의 재조합 아데노바이러스 벡터를 이용하여 Prnp 유전자를 신경 세포주(SH-SY5Y)에 형질 감염한 시스템은 세포 내에서 PrPC의 단백질을 과발현시키고, 일본 뇌염 감염으로 인한 세포내 오토파지를 억제시키며, 일본뇌염 바이러스의 RNA와 단백질 발현 수준을 유의하게 감소시켜, 일본 뇌염 바이러스에 의한 감염증에 치료 효과가 있다. The system in which the Prnp gene is transfected into a neural cell line (SH-SY5Y) using the recombinant adenovirus vector of the present invention overexpresses the PrP C protein within the cell and inhibits intracellular autophagy caused by Japanese encephalitis infection. It has a therapeutic effect on infections caused by Japanese encephalitis virus by significantly reducing the RNA and protein expression levels of the Japanese encephalitis virus.
도 1은 JEV의 감염이 신경 세포를 사멸시킨 것을 확인한 것으로, (A)는 SY5Y 세포를 5 MOI에서 JEV로 감염 시킨 후 광학 현미경으로 확인한 결과이고, (B)는 상기 세포를 크리스탈 바이올렛으로 염색한 결과이며, (C)는 크리스탈 바이올렛 염색을 대조군 세포의 생존율을 100%로 설정하고, 상대 생존율을 계산하여 정량적으로 확인한 결과이고, (D)는 트립판 블루 염색을 통해 세포 생존율을 확인한 결과이다.
도 2는 JEV 감염에 따른 PrPC의 감소를 확인한 것으로, (A) 및 (B)는 JEV 감염 시간에 따른 NS3의 단백질 발현 수준 및 PrPc의 단백질 발현 수준을 각각 확인한 결과이고, (C) 및 (D)는 JEV 감염 다중도에 따른 NS3의 단백질 발현 수준 및 PrPC의 단백질 발현 수준을 확인한 결과이며, (E) 및 (F)는 세포 및 배양 배지에서 JEV 감염 다중도에 따른 JEV RNA 수준을 확인한 결과이다.
도 3은 PrPC의 고갈에 따른 JEV 감염에 따른 신경 세포의 사멸의 촉진을 확인한 것으로서, (A)는 ZW 13-2 (Prnp+/+) 세포 및 Zpl 3-4 (Prnp-/-)를 JEV (5.0 MOI)를 감염시킨 뒤 이를 광학 현미경에서 확인한 결과이고, (B)는 이를 크리스탈 바이올렛으로 염색한 결과이며, (C)는 상기 크리스탈 바이올렛을 결과를 대조군 세포의 생존율을 100%로 설정하고 상대 생존율을 계산한 결과이고, (D)는 ZW 13-2 (Prnp+/+) 세포 및 Zpl 3-4 (Prnp-/-)를 JEV (5.0 MOI)를 감염시킨 세포에서 얻은 세포 배양 상층액으로 부터 LDH의 농도를 측정한 결과이다.
도 4는 PrPC의 고갈에 따른 JEV의 복제를 확인한 것으로서, (A) 및 (B)는 24시간 동안 ZW 13-2 (Prnp+/+) 세포 및 Zpl 3-4 (Prnp-/-)를 JEV (5.0 MOI)를 감염시킨 뒤, 이의 세포에서 NS3 및 PrPC의 단백질 발현 수준을 확인한 결과이고, (C) 및 (D)는 상기 세포에서 JEV 및 Prnp의 RNA 수준을 확인한 결과이며, (E) 및 (F)는 ZW 13-2 (Prnp+/+) 세포 및 Zpl 3-4 (Prnp-/-)를 JEV를 감염시킨 뒤, 시간대 별로 NS3 및 PrPC의 발현을 확인한 결과이다.
도 5는 PrPC의 과발현에 따른 JEV 복제를 확인한 것으로서, (A)는 AD-Prnp 또는 AD-empty 형질감염된 SH-SY5Y 세포를 24시간 동안 JEV (5.0 MOI)를 감염시킨 뒤 이의 JEV mRNA 발현 수준을 확인한 결과이고, (B) 및 (C)는 상기 세포의 PrPC 및 NS3의 단백질 발현 수준을 확인한 결과이고, (D)는 상기 NS3의 단백질 발현 수준을 정량화한 결과이다.
도 6은 PrPc 결핍에 따른 JEV의 생상적인 내재화를 확인한 결과로서, (A)는 SH-SY5Y 세포를 JEV와 함께 배양 후, proteinase K 또는 PBS로 45분 동안 처리한 후, JEV의 RNA 발현을 확인한 결과이고, (B) 및 (C)는 상기 세포의 NS3 및 PrPC의 단백질 발현 수준을 확인한 결과이며, (D)는 SH-SY5Y 세포를 4°C에서 1시간 동안 JEV(MOI=5)로 전 처리한 후 이의 감염된 세포 바이러스의 RNA를 발현 수준을 qPCR로 정량화한 결과이며, (E) 및 (F)은 SH-SY5Y 세포를 37°C에서 1시간 동안 억제제 또는 DMSO로 전처리하고 JEV(MOI=5)를 감염시킨 후, 1시간 동안 4°C로 옮긴 후, 37°C에서 2시간 동안 유지한 다음 이의 세포에서 내부 비리온(internalized virions )의 단백질 발현 및 mRNA 발현 수준을 확인한 결과이고, (G) 및 (H)는 이의 PrPC 및 NS3 단백질 발현 수준을 확인한 결과이다.
도 7은 PrPC의 과발현에 의한 오토파지 유도를 확인한 결과로서, (A) 및 (B)는 SH-SY5Y 세포를 5.0 MOI의 JEV로 24시간 동안 처리한 후 이의 SQSTM1/p62 및 LC3-II 단백질 발현 수준을 확인 결과이고, (C) 및 (D)는 AD-Prnp 또는 AD-empty 형질 감염된 SH-SY5Y 세포를 24시간 동안 5 MOI의 JEV에 감염시킨 후, 이의 SQSTM1/p62 및 LC3-II 단백질 발현 수준을 확인 결과이다.
도 8은 PrPC 결핍에 따른 JEV로 인한 오토파직의 억제를 확인한 것으로, (A) 및 (B)는 ZW 13-2 및 Zp1 3-4 세포를 24시간 동안 5.0 MOI의 JEV로 처리한 후, 이의 SQSTM1/p62 및 LC3-II 단백질 발현 수준을 확인한 결과이고, (C) 및 (D)는 ZW 13-2 및 Zp1 3-4 세 포5.0 MOI의 JEV로 처리한 후 이의 시간대 별로 SQSTM1/p62 및 LC3-II 단백질 발현 수준을 확인한 결과이다.Figure 1 confirms that infection with JEV led to the death of nerve cells. (A) is the result confirmed by light microscopy after infection of SY5Y cells with JEV at 5 MOI, and (B) is the result of staining the cells with crystal violet. This is the result, (C) is the result of quantitative confirmation by setting the survival rate of control cells through crystal violet staining to 100% and calculating the relative survival rate, and (D) is the result of confirming the cell viability through trypan blue staining.
Figure 2 confirms the decrease in PrP C due to JEV infection, (A) and (B) are the results of confirming the protein expression level of NS3 and PrPc according to the JEV infection time, respectively, (C) and ( D) is the result of confirming the protein expression level of NS3 and PrP C according to the multiplicity of JEV infection, and (E) and (F) are the results of confirming the level of JEV RNA according to the multiplicity of JEV infection in cells and culture medium. It is a result.
Figure 3 confirms the acceleration of neuronal death following JEV infection due to depletion of PrP C , (A) shows ZW 13-2 (Prnp +/+ ) cells and Zpl 3-4 (Prnp -/- ) cells. This is the result confirmed under an optical microscope after infection with JEV (5.0 MOI), (B) is the result of staining with crystal violet, and (C) is the result of crystal violet, with the survival rate of control cells set to 100%. This is the result of calculating the relative survival rate, and (D) is the cell culture supernatant obtained from ZW 13-2 (Prnp +/+ ) cells and Zpl 3-4 (Prnp -/- ) cells infected with JEV (5.0 MOI). This is the result of measuring the concentration of LDH.
Figure 4 confirms the replication of JEV following depletion of PrP C. (A) and (B) show ZW 13-2 (Prnp +/+ ) cells and Zpl 3-4 (Prnp -/- ) cells for 24 hours. After infection with JEV (5.0 MOI), the protein expression levels of NS3 and PrP C were confirmed in the cells, (C) and (D) are the results of confirming the RNA levels of JEV and PrPnp in the cells, (E ) and (F) are the results of confirming the expression of NS3 and PrP C at different times after infecting ZW 13-2 (Prnp +/+ ) cells and Zpl 3-4 (Prnp -/- ) cells with JEV.
Figure 5 confirms JEV replication due to overexpression of PrP C. (A) shows the JEV mRNA expression level after infecting AD-Prnp or AD-empty transfected SH-SY5Y cells with JEV (5.0 MOI) for 24 hours. (B) and (C) are the results of confirming the protein expression levels of PrP C and NS3 in the cells, and (D) are the results of quantifying the protein expression levels of NS3.
Figure 6 shows the results confirming the biological internalization of JEV due to PrPc deficiency. (A) shows RNA expression of JEV after culturing SH-SY5Y cells with JEV and treating them with proteinase K or PBS for 45 minutes. These are the results, (B) and (C) are the results confirming the protein expression levels of NS3 and PrP C in the cells, and (D) is the result of inoculating SH-SY5Y cells with JEV (MOI=5) for 1 hour at 4°C. This is the result of quantifying the expression level of virus RNA in infected cells by qPCR after pretreatment. (E) and (F) show SH-SY5Y cells were pretreated with inhibitors or DMSO for 1 hour at 37°C, and JEV (MOI =5), moved to 4°C for 1 hour, maintained at 37°C for 2 hours, and then checked for protein and mRNA expression levels of internalized virions in the cells. (G) and (H) are the results of confirming the PrP C and NS3 protein expression levels.
Figure 7 shows the results confirming the induction of autophagy by overexpression of PrP C. (A) and (B) show the SQSTM1/p62 and LC3-II proteins in SH-SY5Y cells after treatment with JEV at 5.0 MOI for 24 hours. The expression level is confirmed, (C) and (D) show AD-Prnp or AD-empty transfected SH-SY5Y cells were infected with 5 MOI of JEV for 24 hours, and then their SQSTM1/p62 and LC3-II proteins This is the result of confirming the expression level.
Figure 8 confirms the inhibition of autophagy caused by JEV due to PrP C deficiency. (A) and (B) show ZW 13-2 and Zp1 3-4 cells treated with JEV at 5.0 MOI for 24 hours; This is the result of confirming the protein expression levels of SQSTM1/p62 and LC3-II, and (C) and (D) show the expression of SQSTM1/p62 and LC3-II at different time points in ZW 13-2 and Zp1 3-4 cells after treatment with JEV at 5.0 MOI. This is the result of confirming the LC3-II protein expression level.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 재조합 아데노바이러스 벡터를 이용한 PrPc 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방, 진단 또는 치료용 약학적 조성물을 제공할 수 있다. The present invention can provide a pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection containing PrPc protein as an active ingredient using a recombinant adenovirus vector.
본 발명에서 일본 뇌염 바이러스에 감염된 신경 세포가 사멸되는 것을 광학현미경, 크리스탈 바이올렛, 트립판 블루 등의 방법을 사용하여 확인하였다 (도 1A 내지 도 1D). 본 발명자들은 일본 뇌염 바이러스의 치료제를 찾기 위해, 일본 뇌염 바이러스의 감염된 신경 세포에서 특이한 양상을 보이는 유전자를 연구하였다. In the present invention, death of nerve cells infected with Japanese encephalitis virus was confirmed using methods such as light microscopy, crystal violet, and trypan blue (FIGS. 1A to 1D). In order to find a treatment for Japanese encephalitis virus, the present inventors studied genes showing unique patterns in Japanese encephalitis virus-infected nerve cells.
신경 세포인 SH-SY5Y 세포에 JEV를 감염시키면, 시간 의존적으로 JEV NS3가 증가하는 반면, PrPC의 단백질 수준은 감소하는 것을 확인하였다 (도 2A 및 도 2B). 신경 세포인 SH-SY5Y 세포에 JEV의 농도 의존적으로 감염시키면, 농도 의존적으로 JEV NS3가 증가하는 반면, PrPC의 단백질 수준은 감소하는 것을 확인하였다 (도 2A 및 도 2B). JEV RNA는 또한 세포와 배지에서 증가하였다 (도 2E 및 2F). 상기 데이터를 종합적으로 분석한 결과, JEV가 뉴런 세포 사멸 동안 PrPC를 분해하는 것으로 확인하였다. When SH-SY5Y neural cells were infected with JEV, it was confirmed that JEV NS3 increased in a time-dependent manner, while the protein level of PrP C decreased (Figures 2A and 2B). When neuronal SH-SY5Y cells were infected with JEV in a concentration-dependent manner, it was confirmed that JEV NS3 increased in a concentration-dependent manner, while the protein level of PrP C decreased (Figures 2A and 2B). JEV RNA also increased in cells and medium (Figures 2E and 2F). As a result of comprehensive analysis of the above data, it was confirmed that JEV degrades PrP C during neuronal cell death.
이에 Prnp의 발현에 차이가 있는 ZW 13-2 (Prnp+/+) 및 Zpl 3-4 Prnp-/- 마우스 해마 유래 세포를 이용하여 Prnp의 발현에 따른 JEV에 의한 신경 세포 사멸 효과를 확인하였다. 상기 ZW 13-2 (Prnp+/+) 및 Zpl 3-4 Prnp-/- 에 JEV를 감염시킨 후, 광학 현미강, 크리스탈 바이올렛, 트립판 블루를 이용하여 세포 생존율을 확인한 결과 ZW 13-2 (Prnp+/+) 세포보다 Zpl 3-4 (Prnp-/-)세포에서 대조군보다 더 시간 의존적으로 감소하는 것을 확인하였다 (도 3A 내지 도 3C). PrPc의 고갈에 따라서 JEV에 의한 세포 사멸이 촉진되는 것을 확인하였다.Accordingly, the neuronal cell death effect caused by JEV according to the expression of Prnp was confirmed using ZW 13-2 (Prnp +/+ ) and Zpl 3-4 Prnp -/- mouse hippocampus-derived cells, which differ in the expression of Prnp. After infecting the ZW 13-2 (Prnp +/+ ) and Zpl 3-4 Prnp -/- with JEV, cell viability was confirmed using optical microscopy, crystal violet, and trypan blue. As a result, ZW 13-2 ( It was confirmed that the decrease was more time-dependent in Zpl 3-4 (Prnp -/- ) cells than in Prnp +/+ ) cells than in the control group (Figures 3A to 3C). It was confirmed that cell death by JEV was promoted by depletion of PrPc.
상기 Prnp의 발현에 차이가 있는 신경 세포(ZW 13-2 (Prnp+/+) 또는 Zpl 3-4 Prnp-/-)에 JEV를 감염시킨 후, 이의 NS3 및 PrPC 의 단백질 발현 수준을 확인한 결과, JEV 감염이 ZW 13-2 세포에서보다 Zp1 3-4 세포에서 NS3의 세포내 바이러스 단백질 발현이 더 많이 발생한다는 것을 확인하였다 (도 4A). JEV 감염은 ZW 13-2 세포에 존재하는 프리온 단백질의 양을 감소 시켰다 (도 4B). ZW 13-2 (Prnp+/+) 및 Zpl 3-4 (Prnp-/-)를 24시간 동안 5 MOI의 JEV로 처리한 뒤, 실시예 1의 기재된 방법으로 RT-PCR을 분석한 결과, JEV RNA의 양은 ZW 13-2 세포와 비교할 때 Zp1 3-4 세포에서도 증가했으며(도 4C), JEV 감염은 ZW 13-2 세포에서 PrnP mRNA 수준을 감소시켰다 (도 4D). JEV 감염은 ZW 13-2 및 Zp1 3-4세포에서 24시간 후 NS3의 세포내 바이러스 단백질 발현을 극적으로 증가시키는 것을 관찰하였다 (도 4E). JEV 감염 시간에 따라 ZW 13-2 세포에 존재하는 프리온 단백질의 양을 감소하였다 (도 4F). PrPC 발현이 JEV 감염된 해마 뉴런 세포 사멸에서 중요한 역할을 한다는 것을 확인하였다. After infecting nerve cells with differences in the expression of Prnp (ZW 13-2 (Prnp +/+ ) or Zpl 3-4 Prnp -/- ) with JEV, the protein expression levels of NS3 and PrP C were confirmed. , we confirmed that JEV infection resulted in greater intracellular viral protein expression of NS3 in Zp1 3-4 cells than in ZW 13-2 cells (Figure 4A). JEV infection reduced the amount of prion protein present in ZW 13-2 cells (Figure 4B). ZW 13-2 (Prnp +/+ ) and Zpl 3-4 (Prnp -/- ) were treated with 5 MOI of JEV for 24 hours, and then analyzed by RT-PCR using the method described in Example 1. As a result, JEV The amount of RNA was also increased in Zp1 3-4 cells compared with ZW 13-2 cells (Figure 4C), and JEV infection reduced PrnP mRNA levels in ZW 13-2 cells (Figure 4D). We observed that JEV infection dramatically increased the intracellular viral protein expression of NS3 in ZW 13-2 and Zp1 3-4 cells after 24 hours (Figure 4E). The amount of prion protein present in ZW 13-2 cells decreased depending on the JEV infection time (Figure 4F). It was confirmed that PrP C expression plays an important role in JEV-infected hippocampal neuron cell death.
이에 본 발명자들은 아데노 바이러스 벡터에 Prnp 유전자를 형질 전환하여, JEV 감염 조건에서 SH-SY5Y 세포에서 Prnp 유전자를 과발현 하였다. PrPC의 과발현이 JEV 감염을 예방하는지 여부를 확인하기 위해, Ad-PrnP를 형질 감염한 세포에서 JEV의 mRNA의 발현을 RT-PCR 방법으로 확인한 결과, JEV mRNA 수준에서 감소하였다 (도 5A). Ad-PrnP를 형질 감염한 SH-SY5Y 세포는 Ad-empty 형질 감염된 세포와 비교하여 JEV 감염하에 PrPC의 과발현 하는 것을 확인 하였고(도 5B), d-Prnp의 형질 감염은 JEV 감염에 대한 반응에서 NS3의 세포내 바이러스 단백질 발현을 억제하였다 (도 5C 및 도 5D). 즉, PrPC의 과발현이 JEV 감염에 대한 보호 역할을 한다는 것을 확인한 것이다. Accordingly, the present inventors transformed the Prnp gene into an adenovirus vector and overexpressed the Prnp gene in SH-SY5Y cells under JEV infection conditions. To determine whether overexpression of PrP C prevents JEV infection, the expression of JEV mRNA in cells transfected with Ad-PrnP was confirmed by RT-PCR, and the results showed a decrease in the level of JEV mRNA (Figure 5A). SH-SY5Y cells transfected with Ad-PrnP were confirmed to overexpress PrP C under JEV infection compared with Ad-empty transfected cells (Figure 5B), and transfection of d-PrnP was found to be effective in response to JEV infection. Inhibited intracellular viral protein expression of NS3 (Figure 5C and Figure 5D). In other words, it was confirmed that overexpression of PrP C plays a protective role against JEV infection.
PrPc의 결핍에 따른 JEV 감염 경로를 확인한 결과, JEV의 감염은 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 진입 바이러스의 양을 증가 시키고 (도 6A), JEV 감염이 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 NS3 단백질 발현을 증가시켰고, PrPC을 감소하는 것을 확인하였다 (도 6B 및 도 6C). JEV 감염은 또한 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 결합 바이러스의 양을 더 많이 증가시켰다 (도 6D). 그러나 ZW 13-2와 Zp1 3-4 세포 간에 JEV 내재화량의 차이는 관찰되지 않았다 (도 6E 및 도 6F). ZW 13-2 및 Zp1 3-4 세포 사이에 결합 NS3 단백질 수준의 유의한 차이는 관찰되지 않았다 (도 6G). JEV 처리는 ZW 13-2 세포에서 결합 프리온 단백질 수준을 증가시키고 내부 프리온 단백질 수준을 감소시켰고 (도 6H), 이는 바이러스 내재화가 프리온 단백질의 존재에 의해 영향을 받지 않음을 시사하는 결과이다. As a result of confirming the JEV infection route following PrPc deficiency, JEV infection increased the amount of entering virus in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp -/- ) cells (Figure 6A), it was confirmed that JEV infection increased NS3 protein expression and decreased PrP C in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp -/- ) cells (Figures 6B and Figure 6C). JEV infection also increased the amount of bound virus to a greater extent in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp −/− ) cells (Figure 6D). However, no difference in the amount of JEV internalization was observed between ZW 13-2 and Zp1 3-4 cells (Figures 6E and 6F). No significant differences in bound NS3 protein levels were observed between ZW 13-2 and Zp1 3-4 cells (Figure 6G). JEV treatment increased bound prion protein levels and decreased internal prion protein levels in ZW 13-2 cells (Figure 6H), a result suggesting that virus internalization is not affected by the presence of prion protein.
상기 PrPc 단백질은 Prnp 유전자를 재조합 아데노바이러스 벡터에 삽입하여 발현할 수 있으며, 상기 Prnp 유전자는 NCBI Accession No.NM_000311, version NM_000311.3일 수 있으나, 이에 한정되는 것은 아니다. 상기 Prnp 유전자를 아데노 바이러스 벡터에 삽입할 경우 정상 산소 상태에서 PrPC의 단백질의 발현을 증가시키는 것을 특징으로 할 수 있다. The PrPc protein can be expressed by inserting the Prnp gene into a recombinant adenovirus vector, and the Prnp gene may be NCBI Accession No.NM_000311, version NM_000311.3, but is not limited thereto. When the Prnp gene is inserted into an adenovirus vector, the expression of the PrP C protein can be increased under normal oxygen conditions.
상기 조성물은 일본 뇌염 바이러스 감염으로 인한 자가포식(autophage)을 억제하는 것을 특징으로 할 수 있다. The composition may be characterized by inhibiting autophagy caused by Japanese encephalitis virus infection.
SH-SY5Y 세포에서 JEV 감염은 존재하는 SQSTM1/p62 단백질의 양을 감소시켰지만 시간 의존적으로 존재하는 LC3-II의 양을 증가 되었다가 다시 감소하는 것을 확인하였다 (도 7A 및 도 7B). SH-SY5Y 세포에서 PrPC의 과발현 한 실험군은 SQSTM1/p62의 단백질 양은 증가하였고,LC3-II의 단백질 수준도 증가하는 것을 확인할 수 있었다 (도 7C 및 도 7D). 이는 JEV 감염에 의한 오토파지의 활성화가 PrPC의 과발현에 의해 억제 되는 것을 확인하였다. In SH-SY5Y cells, JEV infection decreased the amount of SQSTM1/p62 protein present, but the amount of LC3-II present was confirmed to increase and then decrease again in a time-dependent manner (Figures 7A and 7B). In the experimental group that overexpressed PrP C in SH-SY5Y cells, it was confirmed that the protein level of SQSTM1/p62 increased and the protein level of LC3-II also increased (Figures 7C and 7D). This confirmed that the activation of autophagy due to JEV infection was suppressed by overexpression of PrP C.
어 있는 웨스턴 블롯 분석에 의해 SQSTM1/p62 및 LC3-II 생산에 대해 평가하였다. JEV 감염이 존재하는 SQSTM1/p62 단백질의 양을 감소시키고 ZW 13-2 및 Zp1 3-4 세포에서 LC3-II의 양을 증가시키는 것을 확인하였다 (도 8A 및 B). JEV 감염 6시간 동안 ZW 13-2 및 Zp1 3-4 세포에서 SQSTM1/p62 단백질의 양이 증가하였고, 그 후에 양이 감소시켰다 (도 8C). JEV 처리는 ZW 13-2 (Prnp+/+) 세포에서보다 Zpl 3-4 (Prnp-/-) 세포에 존재하는 LC3-II의 양을 더 많이 증가하였다(도 8D). 이러한 결과는 JEV 감염 조건에서 프리온 단백질의 분해에 의한 오토파지 활성화를 보여준다. SQSTM1/p62 and LC3-II production were evaluated by Western blot analysis. It was confirmed that JEV infection decreased the amount of SQSTM1/p62 protein present and increased the amount of LC3-II in ZW 13-2 and Zp1 3-4 cells (Figures 8A and B). The amount of SQSTM1/p62 protein increased in ZW 13-2 and Zp1 3-4 cells during 6 hours of JEV infection, and then decreased (Figure 8C). JEV treatment increased the amount of LC3-II present in Zpl 3-4 (Prnp − / − ) cells to a greater extent than in ZW 13-2 (Prnp +/+ ) cells (Figure 8D). These results show autophagy activation by decomposition of prion protein under JEV infection conditions.
본 발명의 조성물은 재조합 아데노바이러스 벡터를 이용한 Prnp 유전자를 유효성분으로 포함하는 일본 뇌염 바이러스 감염증의 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may contain one or more known active ingredients that have a preventive or therapeutic effect on Japanese encephalitis virus infection, including the Prnp gene using a recombinant adenovirus vector as an active ingredient.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약학적으로 허용가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.For administration, the composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above. Pharmaceutically acceptable carriers can be saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients, and if necessary, antioxidants and buffer solutions. Other common additives such as bacteriostatic agents can be added. Additionally, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 상기 재조합 아데노바이러스 벡터를 이용한 Ｐｒｎｐ 유전자의 일일 투여량은 약 1~1000 MOI(multiplicity of infection), 바람직하게는 약 50~500 MOI 이나 임상 실험 결과에 따라 가감될 수 있으며, 하루 일회 내지 수회에 나누어 투여하는 것이 바람직하다.The composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage is determined by the patient's weight, age, gender, and health status. , the range varies depending on diet, administration time, administration method, excretion rate, and severity of disease. The daily dose of the PRP gene using the recombinant adenovirus vector is about 1 to 1,000 MOI (multiplicity of infection), preferably about 50 to 500 MOI, but can be adjusted depending on the results of clinical trials, and is administered once or several times a day. It is desirable to administer
본 발명의 조성물은 일본 뇌염 바이러스 감염증의 예방 또는 치료 효과를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response regulators to prevent or treat Japanese encephalitis virus infection.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.below, The present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.
실시예 1. 일본 뇌염 바이러스에 감염된 신경 세포에서 사멸 유도의 확인Example 1. Confirmation of death induction in nerve cells infected with Japanese encephalitis virus
일본 뇌염 바이러스에 의한 신경 세포 사멸의 유도를 조사하였다. SH-SY5Y는 American Type Culture Collection (ATCC, Rockville, MD, USA)에서 구입하였다. SH-SY5Y는 최소 필수 배지(MEM; HyClone Laboratories, Logan, UT, USA)에서 37°C 및 5% CO2 의 조건에서 배양되었다. SH-SY5Y 세포는 5.0의 감염 다중도(MOI)로 JEV(Japanese encephalitis virus; 일본 뇌염 감염 바이러스)를 감염 시켰다. 광학 현미경으로 촬영한 결과 JEV에 감염된 세포가 대조군에 비하여 시간에 따라서 점차 감소하는 것을 확인하였다 (도 1A). The induction of neuronal cell death by Japanese encephalitis virus was investigated. SH-SY5Y was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). SH-SY5Y was cultured in minimal essential medium (MEM; HyClone Laboratories, Logan, UT, USA) at 37°C and 5% CO2 . SH-SY5Y cells were infected with JEV (Japanese encephalitis virus) at a multiplicity of infection (MOI) of 5.0. As a result of imaging with an optical microscope, it was confirmed that the number of JEV-infected cells gradually decreased over time compared to the control group (Figure 1A).
세포의 형태 (Cell morphology)는 광학 현미경으로 확인하였지만, 정확한 세포 생존력을 확인하기 위해 크리스탈 바이올렛 염색(C0775, Sigma-Aldrich, St. Louis, MO, USA)을 수행하였다. 세포를 크리스탈 바이올렛 용액 (30% 에탄올 및 3% 포름알데히드에 0.5% 크리스탈 바이올렛)을 이용하여 실온에서 10분 동안 염색하고 물을 이용하여 5회 세척한 후 건조하였다. 이어서 1% SDS(sodium dodecyl sulphate)로 세포를 용해하고 550nm에서 흡광도를 측정하였다. 대조군과 비교한 샘플의 상대적인 염색 강도로부터 세포 생존율을 계산하였다. 그 결과 광학 현미경에서 관찰 된 것처럼, 크리스탈 바이올렛 분석결과도 JEV에 감염된 세포가 대조군에 비하여 시간에 따라서 점차 감소하였다 (도1 B 및 도 1C).Cell morphology was confirmed by light microscopy, but crystal violet staining (C0775, Sigma-Aldrich, St. Louis, MO, USA) was performed to confirm accurate cell viability. Cells were stained with crystal violet solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde) at room temperature for 10 minutes, washed five times with water, and dried. Then, cells were lysed with 1% SDS (sodium dodecyl sulphate) and absorbance was measured at 550 nm. Cell viability was calculated from the relative staining intensity of the samples compared to the control. As a result, as observed under an optical microscope, the crystal violet analysis results showed that the number of JEV-infected cells gradually decreased over time compared to the control group (Figure 1B and Figure 1C).
생존 세포의 수는 트립판 블루 염료 (Sigma-Aldrich)로 염색한 후, 혈구 계산기(hemocytometer)를 이용하여 측정하였다. 대조군에 대한 백분율로 표시하였다. 트립판 블루 염색을 통해 혈구 계산기를 측정한 결과, 상기와 같이 시간에 따라서 점차 감소되는 것으로 확인 되었다 (도 1D). The number of viable cells was measured using a hemocytometer after staining with trypan blue dye (Sigma-Aldrich). Expressed as a percentage relative to the control group. As a result of measuring the blood count through trypan blue staining, it was confirmed that the blood flow gradually decreased over time as described above (Figure 1D).
실시예 2. 일본 뇌염 바이러스에 감염된 신경 세포에서 PrPExample 2. PrP in neurons infected with Japanese encephalitis virus CC 의 발현 확인Confirmation of expression of
웨스턴 블랏 분석(Western blot analysis)와 실시간 PCR (real-time PCR)을 이용하여 JEV가 신경 세포에서 프리온 단백질에 미치는 영향을 조사하였다. SH-SY5Y 세포를 2.5, 5, 10 MOI의 JEV로 24시간 동안 처리한 후, 처리된 세포를 웨스턴 블롯 분석을 이용하여 3 및 프리온 단백질 수준에 대해 평가하였다. SH-SY5Y 세포를 2.5, 5, 10 MOI의 JEV로 24시간 동안 처리한 후, SH-SY5Y 세포 용해 버퍼 (25 mM HEPES; pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.1mMDTT및 프로테아제 저해 혼합물 (protease inhibitor mixture))로 용해시켰다. 단백질들을 10-15% SDS (소디움 도데실 설페이트 (SDS)) 겔 위에 용해시키고, 공지된 방법으로 면역 블롯팅을 수행하였다. 용해 단백질의 동일 양을 10-15% SDS 폴리아크릴아미드 겔 위에 용해시키고, 니트로셀룰로오즈 멤브레인으로 이동시켰다. 호스래디쉬 퍼옥시다아제 (horseradish peroxidase)가 접합되어 있는 2차 항체 wereanti-NS3 (GTX125868, GeneTex), PrPC (ab52604, Abcam), anti-P62 (#5114; Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA) 및 anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA) 였다. SH-SY5Y 세포에 JEV를 감염시키면, 시간 의존적으로 JEV NS3가 증가하는 것을 확인하였다 (도 2A). SH-SY5Y 세포에 JEV를 감염시키면 PrPC의 단백질 수준도은 시간에 따라 감소하는 것을 확인할 수 있었다. 또한 SH-SY5Y 세포에 농도 증가적으로 JEV를 감염시키면, 농도 의존적으로 JEV NS3의 단백질 수준은 증가하는 반면에(도 2C). PrPC의 단백질 수준은 JEV 감염 농도에 따라 감소하는 것을 확인하였다 (도 2D). The effect of JEV on prion protein in nerve cells was investigated using Western blot analysis and real-time PCR. After SH-SY5Y cells were treated with JEV at 2.5, 5, and 10 MOI for 24 hours, treated cells were assessed for 3 and prion protein levels using Western blot analysis. SH-SY5Y cells were treated with JEV at 2.5, 5, and 10 MOI for 24 h, then lysed in SH-SY5Y cell lysis buffer (25 mM HEPES; pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl, 0.1mMDTT, and It was dissolved with protease inhibitor mixture. Proteins were dissolved on a 10-15% sodium dodecyl sulfate (SDS) gel, and immunoblotting was performed using a known method. Equal amounts of solubilized protein were solubilized on a 10-15% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. Horseradish peroxidase-conjugated secondary antibodies wereanti-NS3 (GTX125868, GeneTex), PrP C (ab52604, Abcam), anti-P62 (#5114; Cell Signaling Technology), LC3 (Novus Biologicals, Littleton, CO, USA) and anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA). When SH-SY5Y cells were infected with JEV, JEV NS3 was confirmed to increase in a time-dependent manner (Figure 2A). When SH-SY5Y cells were infected with JEV, the protein level of PrP C was confirmed to decrease over time. Additionally, when SH-SY5Y cells were infected with JEV at increasing concentrations, the protein level of JEV NS3 increased in a concentration-dependent manner (Figure 2C). The protein level of PrP C was confirmed to decrease depending on the JEV infection concentration (Figure 2D).
SY5Y 세포를 2.5, 5, 10 MOI의 JEV로 24시간 동안 처리한 후, 처리된 세포의 총 RNA를 Easy-spin™ total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea)를 사용하여 추출하였다. cDNA 합성은 TaKaRa Prime ScriptTM 1 st strand cDNA synthesis kit (Takara Bio Inc.,Tokyo, Japan)를 사용하여 공지된 방법으로 수행하였다. 사용된 프라이머는 하기와 같다.After SY5Y cells were treated with JEV at 2.5, 5, and 10 MOI for 24 hours, total RNA of the treated cells was extracted using Easy-spin™ total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea). cDNA synthesis was performed using a known method using the TaKaRa Prime Script TM 1 st strand cDNA synthesis kit (Takara Bio Inc., Tokyo, Japan). The primers used are as follows.
JEV 정방향 프라이머: 5′CCC TCA GAA CCG TCT CGG AA 3′JEV Forward Primer: 5′CCC TCA GAA CCG TCT CGG AA 3′
JEV 역방향 프라이머: 5′ CTA TTC CCA GGT GTC AAT ATG CTG T 3′JEV Reverse Primer: 5′ CTA TTC CCA GGT GTC AAT ATG CTG T 3′
TNF-α 정방향 프라이머: 5′TCT CCT TCC TGA TCG TGG C′TNF-α forward primer: 5′TCT CCT TCC TGA TCG TGG C′
TNF-α 역방향 프라이머: 5′ GGT TCA GCC ACT GGA GCT 3′TNF-α reverse primer: 5′ GGT TCA GCC ACT GGA GCT 3′
IL-6 정방향 프라이머: 5′ AAA TTC GGT ACA TCC TCG AC 3′ IL-6 forward primer: 5′ AAA TTC GGT ACA TCC TCG AC 3′
IL-6 역방향 프라이머: 5′ CAG GAA CTG GAT CAG GAC TT 3′. IL-6 reverse primer: 5′ CAG GAA CTG GAT CAG GAC TT 3′.
상기 프라이머를 사용하여 실시간 PCR을 실시하였다. Real-time PCR was performed using the above primers.
증폭 반응의 20μl은 1μl의 유전자 특이적 프라이머, SYBR Green Supermix(Bio-Rad Laboratories, Hercules, CA) 및 2 μl cDNA가 포함되어 있었다. 모든 정량적 PCR 반응은 CFX96 실시간 PCR 시스템(Bio-Rad)을 사용하여 처리하였다. JEV RNA는 또한 세포와 배지에서 증가하였다 (도 2E 및 2F). 상기 데이터를 종합적으로 분석한 결과, JEV가 뉴런 세포 사멸 동안 PrPC를 분해하는 것으로 확인하였다. 20 μl of amplification reaction contained 1 μl of gene-specific primers, SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and 2 μl cDNA. All quantitative PCR reactions were processed using the CFX96 real-time PCR system (Bio-Rad). JEV RNA also increased in cells and medium (Figures 2E and 2F). As a result of comprehensive analysis of the above data, it was confirmed that JEV degrades PrP C during neuronal cell death.
실시예 3. PrPc의 고갈에 따른 JEV에 의한 세포 사멸의 촉진 확인Example 3. Confirmation of promotion of cell death by JEV following depletion of PrPc
PrPC 발현이 JEV 감염 신경 세포 사멸에 영향을 미치는지 여부를 조사하였다. 마우스 뉴러 세포인 ZW 13-2 및 Zpl 3-4은 ICR (Prnp+/+) 마우스의 및 Zurich I Prnp-/- 마우스의 해마에서 각각 확립되었으며, 김영선 교수님으로부터 제공 받았다. ZW 13-2 및 Zpl 3-4 세포는 10% 소태아혈청(FBS; Invitrogen-GIBCO, Grand Island, NY, USA) 및 젠타마이신(0.1mg)를 포함하는 Dulbecco의 Modified Eagle 배지(DMEM; HyClone Laboratories)에서 37°C 및 5% CO2 의 조건에서 배양되었다. 세포는 5.0의 감염 다중도(MOI)에서 JEV를 감염시켰다. 현미경 사진 결과, ZW 13-2 (Prnp+/+) 세포보다 Zpl 3-4 (Prnp-/-)세포에서 대조군보다 더 시간 의존적으로 세포 생존률이 감소하는 것을 확인하였다 (도 3A).We investigated whether PrPC expression affects JEV-infected neuronal cell death. Mouse neuron cells, ZW 13-2 and Zpl 3-4, were established from the hippocampus of ICR (Prnp +/+ ) mice and Zurich I Prnp −/− mice, respectively, and were provided by Professor Youngsun Kim. ZW 13-2 and Zpl 3-4 cells were grown in Dulbecco's Modified Eagle's medium (DMEM; HyClone Laboratories) containing 10% fetal bovine serum (FBS; Invitrogen-GIBCO, Grand Island, NY, USA) and gentamicin (0.1 mg). ) were cultured under conditions of 37°C and 5% CO 2 . Cells were infected with JEV at a multiplicity of infection (MOI) of 5.0. As a result of the micrograph, it was confirmed that the cell survival rate decreased more in a time-dependent manner in Zpl 3-4 (Prnp -/- ) cells than in the control group than in ZW 13-2 (Prnp +/+ ) cells (Figure 3A).
상기 실시예 1의 방법으로 크리스탈 바이올렛 염색을 이용하여 세포 생존력을 확인한 결과, 세포는 ZW 13-2 (Prnp+/+) 세포와 비교할 때 Zpl 3-4 (Prnp-/-)에서 더 시간 의존적으로 감소하였다 (도 3B 및 도 3C). LDH Cytotoxicity Detection kit (Takara Bio Inc., Tokyo, Japan)를 이용하여 상청액 배지에서 LDH 분석법을 측정하였다. LDH 활성은 490nm 에서 흡광도를 측정하여 결정하였다. LDH 분석에서 신경 세포주 사이에 유의한 차이는 관찰하지 못하였다 (도 3D). 상기 데이트를 종합한 결과 PrPc의 고갈에 따라서 JEV에 의한 세포 사멸이 촉진되는 것을 확인하였다.As a result of confirming cell viability using crystal violet staining using the method of Example 1, cells were more time-dependent in Zpl 3-4 (Prnp -/- ) compared to ZW 13-2 (Prnp +/+ ) cells. decreased (Figure 3B and Figure 3C). LDH assay was measured in the supernatant medium using the LDH Cytotoxicity Detection kit (Takara Bio Inc., Tokyo, Japan). LDH activity was determined by measuring absorbance at 490 nm. We did not observe significant differences between neuronal cell lines in the LDH analysis (Figure 3D). As a result of combining the above data, it was confirmed that cell death by JEV is promoted by depletion of PrPc.
실시예 4. PrPc의 고갈에 따른 JEV의 복제 유도의 확인Example 4. Confirmation of replication induction of JEV following depletion of PrPc
뉴런 세포에서 프리온 단백질을 고갈시키는 JEV의 역할을 확인하기 위해 상기 실시예 1의 방법으로 웨스턴 블롯 분석과 실시간 PCR을 사용 하였다. ZW 13-2 (Prnp+/+) 및 Zpl 3-4 (Prnp-/-)를 24시간 동안 5 MOI의 JEV로 처리한 뒤, 실시예 1의 기재된 방법으로 웨스턴 블랏을 수행하여 이의 NS3 및 프리온의 단백질 발현 수준을 확인하였다. 그 결과 JEV 감염이 ZW 13-2 세포에서보다 Zp1 3-4 세포에서 NS3의 세포내 바이러스 단백질 발현이 더 많이 발생한다는 것을 확인하였다 (도 4A). JEV 감염은 ZW 13-2 세포에 존재하는 프리온 단백질의 양을 감소 시켰다 (도 4B). ZW 13-2 (Prnp+/+) 및 Zpl 3-4 (Prnp-/-)를 24시간 동안 5 MOI의 JEV로 처리한 뒤, 실시예 1의 기재된 방법으로 RT-PCR을 분석한 결과, JEV RNA의 양은 ZW 13-2 세포와 비교할 때 Zp1 3-4 세포에서도 증가했으며(도 4C), JEV 감염은 ZW 13-2 세포에서 PrnP mRNA 수준을 감소시켰다 (도 4D). ZW 13-2 (Prnp+/+) 및 Zpl 3-4 (Prnp-/-) 세포에서 JEV 감염 시간에 따른 NS3 및 PrPC의 단백질 발현 수준을 실시예 1의 기재된 방법으로 측정한 결과, JEV 감염은 ZW 13-2 및 Zp1 3-4세포에서 24시간 후 NS3의 세포내 바이러스 단백질 발현을 극적으로 증가시키는 것을 관찰하였다 (도 4E). JEV 감염 시간에 따라 ZW 13-2 세포에 존재하는 프리온 단백질의 양을 감소하였다 (도 4F). 따라서, PrPC 발현이 JEV 감염된 해마 뉴런 세포 사멸에서 중요한 역할을 한다는 것을 확인하였다. To confirm the role of JEV in depleting prion protein in neuronal cells, Western blot analysis and real-time PCR were used using the method of Example 1 above. ZW 13-2 (Prnp +/+ ) and Zpl 3-4 (Prnp -/- ) were treated with 5 MOI of JEV for 24 hours, and then Western blot was performed by the method described in Example 1 to determine their NS3 and prion expression. The protein expression level was confirmed. As a result, it was confirmed that JEV infection resulted in more intracellular viral protein expression of NS3 in Zp1 3-4 cells than in ZW 13-2 cells (Figure 4A). JEV infection reduced the amount of prion protein present in ZW 13-2 cells (Figure 4B). ZW 13-2 (Prnp +/+ ) and Zpl 3-4 (Prnp -/- ) were treated with 5 MOI of JEV for 24 hours, and as a result of RT-PCR analysis by the method described in Example 1, JEV The amount of RNA was also increased in Zp1 3-4 cells compared with ZW 13-2 cells (Figure 4C), and JEV infection reduced PrnP mRNA levels in ZW 13-2 cells (Figure 4D). As a result of measuring the protein expression levels of NS3 and PrP C in ZW 13-2 (Prnp +/+ ) and Zpl 3-4 (Prnp -/- ) cells according to the JEV infection time by the method described in Example 1, JEV infection observed a dramatic increase in intracellular viral protein expression of NS3 in ZW 13-2 and Zp1 3-4 cells after 24 hours (Figure 4E). The amount of prion protein present in ZW 13-2 cells decreased depending on the JEV infection time (Figure 4F). Therefore, it was confirmed that PrP C expression plays an important role in JEV-infected hippocampal neuron cell death.
실시예 5. PrPExample 5. PrP CC 의 과발현에 따른 신경 세포에서 JEV 복제의 하향 조절 확인Confirmation of downregulation of JEV replication in neurons following overexpression of
PrPC가 JEV의 복제 조절 효과를 확인하기 위해, 재조합 아데노바이러스 벡터에 Prnp 유전자를 형질 전환하여, JEV 감염 조건에서 SH-SY5Y 세포에서 Prnp 유전자를 과발현 하였다. 인간 전장 Prnp 유전자(NCBI Accession No. NM_000311, version NM_000311.3)를 발현하는 재조합 아데노바이러스 벡터(Ad-Prnp)는 (주)제넨메드(서울, 한국)로부터 제공받았다. 상기 재조합 아데노바이러스 벡터에 삽입된 Prnp 유전자는 정상 산소 상태에서 PrPC의 mRNA 및 단백질의 발현을 증가시키는 것을 이전 연구에서 확인하였다. 전장 PRNP 유전자(Ad-PRNP)를 발현하지 않는 카세트(cassette)인 재조합 바이러스(Ad-empty)를 대조군으로 사용하였다. 세포를 24시간 동안 FBS가 없는 MEM 배지에서 Ad-PRNP 및 Ad-LacZ로 형질전환 하였다. 멸균된 PBS 완충액으로 세척한 후 2% FBS를 포함하는 DMEM을 첨가하였다. 포스트 아데노바이러스 형질 전환 기간 동안 PrPC의 과발현이 JEV 감염을 예방하는지 여부를 확인하기 위해 250 및 500의 감염 다중도에서 Ad-PrnP를 형질 감염한 세포에서 JEV의 mRNA의 발현을 실시예 1의 RT-PCR 방법으로 확인한 결과, 250 및 500의 감염 다중도 모두 JEV mRNA 수준에서 감염이 억제되었다. 그러나 Ad-empty로 형질 감염된 PrPC의 보호 효과는 없었다 (도 5A). PrPC의 과발현을 PrPC 항체를 사용하여 실시예 1에 기재된 웨스턴 블랏을 이용하여 SH-SY5Y 세포에서 확인하였다. Ad-PrnP를 형질 감염한 SH-SY5Y 세포는 Ad-empty 형질 감염된 세포와 비교하여 JEV 감염하에 PrPC의 과발현 하는 것을 확인하였다 (도 5B). 500의 감염 다중도 (MOI)에서 Ad-Prnp의 형질 감염은 JEV 감염에 대한 반응에서 NS3의 세포내 바이러스 단백질 발현을 억제하였다 (도 5C 및 도 5D). 즉, PrPC의 과발현이 JEV 감염에 대한 보호 역할을 한다는 것을 확인한 것이다. To confirm the effect of PrP C on regulating the replication of JEV, the Prnp gene was transformed into a recombinant adenovirus vector and overexpressed in SH-SY5Y cells under JEV infection conditions. A recombinant adenovirus vector (Ad-Prnp) expressing the full-length human Prnp gene (NCBI Accession No. NM_000311, version NM_000311.3) was provided by Genenmed Co., Ltd. (Seoul, Korea). Previous studies have confirmed that the Prnp gene inserted into the recombinant adenovirus vector increases the expression of PrP C mRNA and protein under normal oxygen conditions. A recombinant virus (Ad-empty), which is a cassette that does not express the full-length PRNP gene (Ad-PRNP), was used as a control. Cells were transfected with Ad-PRNP and Ad-LacZ in MEM medium without FBS for 24 hours. After washing with sterilized PBS buffer, DMEM containing 2% FBS was added. To determine whether overexpression of PrP C prevents JEV infection during the post adenoviral transduction period, expression of the mRNA of JEV was measured in cells transfected with Ad-PrnP at a multiplicity of infection of 250 and 500 at RT as in Example 1. -As confirmed by PCR method, infection was suppressed at the JEV mRNA level at both multiplicities of infection of 250 and 500. However, there was no protective effect of PrPC transfected with Ad-empty (Figure 5A). Overexpression of PrP C was confirmed in SH-SY5Y cells using Western blotting as described in Example 1 using PrP C antibody. SH-SY5Y cells transfected with Ad-PrnP were confirmed to overexpress PrP C under JEV infection compared to cells transfected with Ad-empty (Figure 5B). Transfection of Ad-Prnp at a multiplicity of infection (MOI) of 500 inhibited intracellular viral protein expression of NS3 in response to JEV infection (Figure 5C and Figure 5D). In other words, it was confirmed that overexpression of PrP C plays a protective role against JEV infection.
실시예 6. PrPc의 결핍에 따른 JEV 감염 경로에 미치는 영향의 확인Example 6. Confirmation of the effect of PrPc deficiency on the JEV infection route
감염의 초기 단계에는 결합, 세포막 교차, 세포 이동 및 코팅 해제를 포함한 많은 이벤트가 포함되며 이들 중 어느 것이 프리온 단백질의 영향을 받는지 확인하였다. 진입과 관련하여 4°C에서 흡수된 입자의 수는 JEV RNA의 양을 정량화하였다. SH-SY5Y 세포를 JEV와 함께 4°C에서 1시간 동안 인큐베이션하고 결합되지 않은 바이러스를 차가운 PBS로 세척한 후 4°C에서 proteinase K 또는 PBS로 45분 동안 처리하였다. 상기 세포를 TRIzol을 이용하여 RNA를 추출하고, 실시예 1의 방법을 이용하여 RT-PCR을 수행하였다. JHV의 RNA는 대조군 GAPDH를 사용한 qPCR에 의해 측정하였다. JEV의 감염은 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 진입 바이러스의 양을 증가시켰다 (도 6A). The initial stages of infection involve many events, including binding, cell membrane crossing, cell migration and uncoating, and we determined which of these are affected by the prion protein. The number of absorbed particles at 4°C relative to entry quantified the amount of JEV RNA. SH-SY5Y cells were incubated with JEV for 1 h at 4°C, unbound virus was washed with cold PBS, and then treated with proteinase K or PBS for 45 min at 4°C. RNA was extracted from the cells using TRIzol, and RT-PCR was performed using the method of Example 1. RNA of JHV was measured by qPCR using control GAPDH. Infection with JEV increased the amount of entering virus in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp −/− ) cells (Figure 6A).
SH-SY5Y 세포를 JEV와 함께 4°C에서 1시간 동안 인큐베이션하고 결합되지 않은 바이러스를 차가운 PBS로 세척한 후 실시예 1와 같이 웨스턴 블롯을 수행하여, PrPC 및 NS3의 단백질 수준을 측정하였다. JEV 감염이 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 NS3 단백질 발현을 증가시켰고, PrPC을 감소하는 것을 확인하였다 (도 6B 및 도 6C). SH-SY5Y cells were incubated with JEV at 4°C for 1 hour, unbound virus was washed with cold PBS, and Western blot was performed as in Example 1 to measure protein levels of PrP C and NS3. It was confirmed that JEV infection increased NS3 protein expression and decreased PrP C in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp -/- ) cells (Figures 6B and 6C). .
결합 바이러스과 관련하여 4°C에서 흡수된 입자의 수는 JEV RNA의 양을 정량화하여 계산하였다. SH-SY5Y 세포는 4°C에서 1시간 동안 JEV(MOI=5)로 전 처리한 후, 세포를 차가운 PBS로 3회 세척한 다음 TRIzol 시약으로 수집하고 실시예 1와 같이 RT-PRC를 수행하여, 감염된 세포의 바이러스 게놈 RNA를 내부 대조군 GAPDH를 사용하여 qPCR로 정량화 하였다. JEV 감염은 또한 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 결합 바이러스의 양을 더 많이 증가시켰다 (도 6D). The number of particles absorbed at 4°C in relation to bound virus was calculated by quantifying the amount of JEV RNA. SH-SY5Y cells were pretreated with JEV (MOI=5) for 1 hour at 4°C, then cells were washed three times with cold PBS, then collected with TRIzol reagent, and RT-PRC was performed as in Example 1. , viral genomic RNA in infected cells was quantified by qPCR using the internal control GAPDH. JEV infection also increased the amount of bound virus to a greater extent in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp −/− ) cells (Figure 6D).
SH-SY5Y 세포를 37°C에서 1시간 동안 억제제 또는 DMSO로 전처리하고 JEV(MOI=5)를 처리하였다. 그런 다음 4°C에서 1시간 동안 배양하였다. 세포를 37°C에서 2시간 동안 유지한 다음 PBS로 세척하였다. 4°C에서 45분 동안 단백질분해효소 K로 처리한 후, RNA 분리를 위해 세포를 TRIzol에 수집하여, 각각 웨스턴 블롯 및 RT-PCR을 수행하여 내부 비리온(virion; entire virus particle)의 단백질 발현 수준과 mRNA 발현 수준을 확인하였다. ZW 13-2와 Zp1 3-4 세포 간에 JEV 내재화량의 차이는 관찰되지 않았다 (도 6E 및 도 6F). JEV 처리가 Zpl 3-4 (Prnp-/-) 세포에서보다 ZW 13-2 (Prnp+/+) 세포에서 결합 NS3 단백질 수준을 증가시키는 것을 관찰하였다. ZW 13-2 및 Zp1 3-4 세포 사이에 결합 NS3 단백질 수준의 유의한 차이는 관찰되지 않았다 (도 6G). JEV 처리는 ZW 13-2 세포에서 결합 프리온 단백질 수준을 증가시키고 내부 프리온 단백질 수준을 감소시켰고 (도 6H), 이는 바이러스 내재화가 프리온 단백질의 존재에 의해 영향을 받지 않음을 시사하는 결과이다. SH-SY5Y cells were pretreated with inhibitors or DMSO for 1 h at 37°C and treated with JEV (MOI=5). Then, it was incubated at 4°C for 1 hour. Cells were kept at 37°C for 2 hours and then washed with PBS. After treatment with proteinase K for 45 minutes at 4°C, cells were collected in TRIzol for RNA isolation, and Western blot and RT-PCR were performed to determine protein expression of internal virions (entire virus particles), respectively. The level and mRNA expression level were confirmed. No difference in the amount of JEV internalization was observed between ZW 13-2 and Zp1 3-4 cells (Figures 6E and 6F). We observed that JEV treatment increased the level of bound NS3 protein in ZW 13-2 (Prnp +/+ ) cells than in Zpl 3-4 (Prnp −/− ) cells. No significant differences in bound NS3 protein levels were observed between ZW 13-2 and Zp1 3-4 cells (Figure 6G). JEV treatment increased bound prion protein levels and decreased internal prion protein levels in ZW 13-2 cells (Figure 6H), a result suggesting that virus internalization is not affected by the presence of prion protein.
실시예 7. PrPc의 과발현으로 인한 오토파지의 조절 확인Example 7. Confirmation of regulation of autophagy due to overexpression of PrPc
PrPc 발현이 JEV 처리된 신경 세포에서 오토파지 프럭스(flux) 신호 전달에 여향을 미치는지 여부를 조사하였다. SH-SY5Y 세포를 5.0 MOI의 JEV로 24시간 동안 처리한 후, 처리된 세포를 실시예 1의 기재된 방법과 같이 웨스턴 블롯 분석을 이용하여 SQSTM1/p62 및 LC3-II의 단백질 수준을 측정하였다. SH-SY5Y 세포에서 JEV 감염은 존재하는 SQSTM1/p62 단백질의 양을 감소시켰지만 시간 의존적으로 존재하는 LC3-II의 양을 증가 되었다가 다시 감소하는 것을 확인하였다 (도 7A 및 도 7B).We investigated whether PrPc expression affected autophagy flux signaling in JEV-treated neurons. After SH-SY5Y cells were treated with 5.0 MOI of JEV for 24 hours, the treated cells were measured for protein levels of SQSTM1/p62 and LC3-II using Western blot analysis as described in Example 1. In SH-SY5Y cells, JEV infection decreased the amount of SQSTM1/p62 protein present, but the amount of LC3-II present was confirmed to increase and then decrease again in a time-dependent manner (Figures 7A and 7B).
PrPC의 과발현이 JEV 감염에 의한 오토파지의 조절을 확인하기 위해, 250 또는 500의 감염 다중도에서 Ad-PrnP를 형질 감염한 세포에서, 실시예 1의 기재된 방법과 같이 웨스턴 블롯 분석을 이용하여 SQSTM1/p62 및 LC3-II의 단백질 수준을 측정하였다. SH-SY5Y 세포에서 PrPC의 과발현 한 실험군은 SQSTM1/p62의 단백질 양은 증가하였고, LC3-II의 단백질 수준도 증가하는 것을 확인할 수 있었다 (도 7C 및 도 7D). 이는 JEV 감염에 의한 오토파지의 활성화가 PrPC의 과발현에 의해 억제 되는 것을 확인하였다. To confirm that overexpression of PrP C modulates autophagy by JEV infection, Western blot analysis was used as described in Example 1 in cells transfected with Ad-PrnP at a multiplicity of infection of 250 or 500. Protein levels of SQSTM1/p62 and LC3-II were measured. In the experimental group that overexpressed PrP C in SH-SY5Y cells, the protein level of SQSTM1/p62 increased, and the protein level of LC3-II also increased (Figures 7C and 7D). This confirmed that the activation of autophagy due to JEV infection was suppressed by overexpression of PrP C.
실시예 8. PrPc의 결핍에 따른 JEV으로 인한 오토파지의 억제의 확인Example 8. Confirmation of inhibition of autophagy caused by JEV due to PrPc deficiency
ZW 13-2 및 Zp1 3-4 세포를 24시간 동안 5MOI에서 JEV로 처리한 후, 처리된 세포를 실시예 1에 기재되어 있는 웨스턴 블롯 분석에 의해 SQSTM1/p62 및 LC3-II 생산에 대해 평가하였다. JEV 감염이 존재하는 SQSTM1/p62 단백질의 양을 감소시키고 ZW 13-2 및 Zp1 3-4 세포에서 LC3-II의 양을 증가시키는 것을 확인하였다 (도 8A 및 B). JEV 감염 6시간 동안 ZW 13-2 및 Zp1 3-4 세포에서 SQSTM1/p62 단백질의 양이 증가하였고, 그 후에 양이 감소시켰다 (도 8C). JEV 처리는 ZW 13-2 (Prnp+/+) 세포에서보다 Zpl 3-4 (Prnp-/-) 세포에 존재하는 LC3-II의 양을 더 많이 증가하였다(도 8D). 이러한 결과는 JEV 감염 조건에서 프리온 단백질의 분해에 의한 오토파지 활성화를 보여준다. ZW 13-2 and Zp1 3-4 cells were treated with JEV at 5MOI for 24 hours, and then treated cells were assessed for SQSTM1/p62 and LC3-II production by Western blot analysis as described in Example 1. . It was confirmed that JEV infection decreased the amount of SQSTM1/p62 protein present and increased the amount of LC3-II in ZW 13-2 and Zp1 3-4 cells (Figures 8A and B). The amount of SQSTM1/p62 protein increased in ZW 13-2 and Zp1 3-4 cells during 6 hours of JEV infection, and then decreased (Figure 8C). JEV treatment increased the amount of LC3-II present in Zpl 3-4 (Prnp − / − ) cells to a greater extent than in ZW 13-2 (Prnp +/+ ) cells (Figure 8D). These results show autophagy activation by decomposition of prion protein under JEV infection conditions.
Claims (4)
A pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection comprising PrPc protein as an active ingredient using a recombinant adenovirus vector.
The pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection according to claim 1, wherein the PrPc protein is expressed by inserting the Prnp gene into a recombinant adenovirus vector.
The pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection according to claim 1, wherein the composition inhibits autophagy caused by Japanese encephalitis virus infection.
The pharmaceutical composition for preventing, diagnosing or treating Japanese encephalitis virus infection according to claim 1, wherein the composition is formulated in oral, parenteral, inhalation or topical administration form.
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| KR (1) | KR20240051530A (en) |
-
2022
- 2022-10-13 KR KR1020220131307A patent/KR20240051530A/en unknown
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