초록 ▼

연구목표
다제내성 감염병 치료를 위하여, CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 유전체편집 기술이 적용된 나노전달체 기반의 다제내성 감염병의 치료법을 실험적으로 검증 및 개발한다.

연구 결과
가. 항생제 저항성 표적 특이적 sgRNA의 설계, 제작 및 SpCas9 단백질의 정제
① 박테리아 샘플에서의 항생제 표적 저항성 유전자 확인.
② SpCas9 protein과 형광 재조합SpCas9 단백질의 세균배양을 통한

연구목표
다제내성 감염병 치료를 위하여, CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 유전체편집 기술이 적용된 나노전달체 기반의 다제내성 감염병의 치료법을 실험적으로 검증 및 개발한다.

연구 결과
가. 항생제 저항성 표적 특이적 sgRNA의 설계, 제작 및 SpCas9 단백질의 정제
① 박테리아 샘플에서의 항생제 표적 저항성 유전자 확인.
② SpCas9 protein과 형광 재조합SpCas9 단백질의 세균배양을 통한 생산 및 정제.
③ 유전자 편집을 위한, PAM sequence를 고려한 표적 특이적 sgRNA의 제작.
나. 효율적인 유전자 편집을 위한 고밀도의 나노전달 제형을 개발
① 높은 세포 유입과 편집 효율 그리고 장기간 동안의 혈중 내 농도 유지를 고려한 합성고분자 나노전달 제형의 설계.
② CRISPR/Cas9 나노제형의 전달 효율 분석을 위한 형광발현 SpCas9나노복합체의 제작
③ 공초점 현미경을 이용한 박테리아 세포에서의 In vitro 전달 가능성 확인.
고분자를 이용한 CRISPR/Cas9 나노제형의 세균 내 전달 가능성을 확인하였으며, 향후 후속 연구를 통해 나노 제형 개발 조건 확립 및 기능적 측면의 분석을 진행 예정임

Abstract ▼

The wide use of antibiotics is playing a major role in the emergence of resistant bacteria that increase serious health problems. The pathogenecity of bacteria is increased mainly due to the transfer of antibiotic resistance genes by means of by horizontal gene transfer via plasmid conjugation or ot

The wide use of antibiotics is playing a major role in the emergence of resistant bacteria that increase serious health problems. The pathogenecity of bacteria is increased mainly due to the transfer of antibiotic resistance genes by means of by horizontal gene transfer via plasmid conjugation or other mobile genetic elements. Thus, the number of the effective antibiotics to combat these pathogens is rapidly decreasing. Patients suffering from antibiotics resistant bacterial infections are generally at bigger risk of worse clinical results and death, and longer suffering and more expensive hospital stays for treatment. Despite the urgent need for new treatments against antibiotics resistant bacteria, very few new classes of antibiotics has been developed in the past decade. These could put at risk many effective trials in control of an ever-increasing range of bacterial infection. In short, the threat of untreatable infections is getting closer. So, scientists are recognizing a necessity of powerful new therapeutics against bacterial infections.

The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand cleavage in simple sequential steps. Streptococcus pyogens Cas9(SpCas9), an enzyme for cutting target specific oligo nucleotides, is the only component in the Type Ⅱ CRISPR system. Combination of tracrRNA(trans activating RNA) and spacer RNA(crRNA) mixed with Cas9 showed highly effective DNA targeting and double strand break (DSB). To target and cleave the specific sequences with ZFN (Zinc-Finger Nuclease) or TALEN (Transcription activator-like effector nuclease), genetic engineering with thousands of bp for DNA are needed. However, the CRISPR system only needs target specific crRNA sequences that are less than 20 bp. In short, a restriction enzyme that can recognize a new sequence can be created by modification of sgRNA in a one-step process. CRISPR shows highly efficient cleavage.

Now, many researchers are recognizing the CRISPR, a gene-editing system that can modify any target gene as an influential tool on genetic editing. Recently, there are some reports that show the selective deletion of bacteria carrying antibiotic resistance with CRISPR-Cas nucleases. This demonstrated the possibility of application of sequence-specific antibiotics using the RNA-guided nuclease Cas9.

Gene therapy is generally carried out by exogenous nucleotides such as messenger RNA (mRNA), DNA or siRNA. To deliver these genes modified viruses such as retroviruses, lentiviruses, adenoviruses and adeno-associated viruses (AAVs) are usually used with highly advanced technologies. Conventional CRISPR system is also accomplished by vectors or carriers due to the large size and the negative charge of nucleic acids CRISPR components (Cas9, tracrRNA and crRNA sequences). However, several limitations are well known with these viral vectors, including immunogenicity, tropism problem, carcinogenesis and difficulty of carrier production. These vectors have induced limitation of successful clinical trials in gene based therapeutics. On the other hand, non-viral carriers system are reported that has lower immunogenicity than viral carriers. These vectors have the ability to deliver larger genetic elements and are comparatively easier to synthesize than viral vectors as well. Various non viral vectors have been studied to bring therapeutic molecules to their target site. Nevertheless, few of these vectors have been developed for clinical use due to their low delivery efficiency relative to viral vectors. This weakness may be about to change due to the rapid advancement of nanotechnology, which makes better understanding of nanosized new polymers as gene delivery vectors. Moreover, recent developments in nucleic acid chemistry will reduce the problems related to immunogenicity with improved efficiency and stability.

Based on this, we decided our candidate for novel nanoparticle that could carry out sgRNA and Cas9 protein targeting antibiotic resistance gene with additional biological insights into the key-determining steps; cationic polymer-antifouling polymer. So far, there has not been any study about CRISPR based sequence specific antimicrobials combined with nano-particles, which can lead to highly efficient microbial control. This approach will need huge repeated trials and errors for technology development as well as validation. Furthermore, the development of efficient targeting-based treatment strategy for bacterial infections with antibiotic resistance will greatly enhance therapeutic efficacy as well as prevent spread or emergence of other multi drug resistant strains. This might be utilized for treating infections with low antibiotic susceptibility or those that do not respond to conventional antibiotics. Finally, our approach will contribute in improving healthcare and reduce costs for inpatient care and excessive treatment regimens.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 보고서 초록 ... 3
• 요약문 ... 4
• SUMMARY ... 6
• CONTENTS ... 9
• 목차 ... 10
• 제 1 장 연구개발과제의 개요 및 국내외 기술개발 현황 ... 11
• 제 2 장 연구개발수행 내용 및 결과 ... 14
• 1절 연구개발 수행 내용 및 결과 ... 14
• 제 3 장 목표달성도 및 관련분야에의 기여도 ... 20
• 가. 연구개발목표의 달성 결과에 대한 설명 ... 20
• 나. 관련분야 기술발전에의 기여도 ... 22
• 제 4 장 연구개발 결과 기반의 추후 계획 ... 23
• 제1절 추가연구의 필요성 ... 23
• 제2절 타 연구에의 응용 ... 24
• 제 5 장 연구개발과정에서 수집한 해외과학기술정보 ... 24
• 제 6 장 References ... 25
• 끝페이지 ... 26