KR20190034239A - 변이체 캡시드를 갖는 아데노-관련된 바이러스 비리온 및 이의 사용 방법 - Google Patents
변이체 캡시드를 갖는 아데노-관련된 바이러스 비리온 및 이의 사용 방법 Download PDFInfo
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Abstract
본 개시내용은 변경된 캡시드 단백질을 갖는 아데노-관련된 바이러스 (AAV) 비리온을 제공하되, 상기 AAV 비리온을 야생형 AAV와 비교하여 망막 세포의 더 큰 감염성을 나타낸다. 본 개시내용은 또한, 개체에서 유전자 산물을 망막 세포에 전달하는 방법, 및 안구 질환을 치료하는 방법을 제공한다.
Description
교차-참조
본원은 미국 특허 가출원 번호 62/368,929(2016년 7월 29일 출원)의 이점을 주장하며, 상기 공보는 본 명세서에 참고로 전체적으로 편입되어 있다.
연방 지원된 연구에 관한 서술
본 발명은 국립 보건원에 의해 수여된 계약/수여 번호 EY022975, EY018241 및 EY06855 하에 정부 지원으로 만들어졌다. 정부는 본 발명에 대해 특정 권리는 갖는다.
광수용체는, 광변조를 통해 가시 전자기 복사를 과분극된 응답으로 전환시키는, 시각 정보를 수용 및 처리하기 위한 망막내 제1 뉴런이다. 유전성 망막 질환의 압도적 다수는 로돕신 단백질 폴딩에 영향을 미치는 우세한 돌연변이처럼 직접적으로, 또는 망막 색소 상피(RPE)의 망막 재활용 경로에 영향을 미치는 열성 돌연변이처럼 간접적으로 이러한 세포들의 손실을 초래한다.
아데노-관련 바이러스(AAV)는 파보비리다애 과 및 데펜도바이러스 속에 속하며, 그 구성원은 복제를 촉진시키기 위해 아데노바이러스와 같은 헬퍼 바이러스와 함께 공-감염되어야 하며, AAV는 헬퍼의 부재 하에서 잠복성 감염을 설정한다. 비리온은 2개의 개방형 판독 프레임: rep 및 cap이 있는 4.9 kb 단일-가닥 DNA 게놈을 포괄하는 25 nm 이십면체 캡시드로 구성된다. 비-구조적 rep 유전자는 바이러스 복제에 필수적인 4개의 조절 단백질을 인코딩하고, 반면 cap은 60-mer 캡시드 쉘로 조립되는 3개의 구조 단백질(VP1-3)을 인코딩한다. 이러한 바이러스 캡시드는 세포 표면 수용체 결합, 내포작용, 세포내 교환, 및 핵에서 언패키징을 포함하는 - 바이러스 형질도입의 생물학적 장벽의 다수를 극복하기 위해 AAV 벡터의 능력을 매개한다.
본 개시내용은 재조합 변경된 캡시드 단백질을 갖는 아데노-관련된 바이러스 (AAV) 비리온을 제공하되, 상기 재조합 AAV (rAAV) 비리온은 야생형 AAV와 비교하여 망막 세포의 더 큰 감염성을 나타내고, 그리고 상기 rAAV 비리온은 이종성 핵산을 포함한다. 본 개시내용은 추가로, 개체에서 유전자 산물을 망막 세포에 전달하는 방법, 및 안구 질환을 치료하는 방법을 제공한다. 본 개시내용은 rAAV 비리온을 제공하되, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해, 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 또는 망막 색소 상피에 대한 국제화의 정도와 비교하여, 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 및 망막 색소 상피 중 하나 이상에 대해 적어도 5-배수 증가된 국재화를 나타낸다.
도 1는 친계 AAV와 비교하여 안구 세포 (예를 들어, 망막 세포)의 증가된 감염성을 나타내는 AAV 변이체를 개발하기 위해 사용된 직접 진화 방법의 도식적 묘사이다.
도 2는 녹색 형광 단백질 (GFP)-바코드 작제물을 함유하는 AAV 변이체의 심화 서열분석의 도식적 묘사이다.
도 3은 18-구성원 AAV 변이체 라이브러리에 의해, 신경절 세포 층, 내부 핵 층, 광수용체 층, 및 망막 색소 상피 (RPE) 층에서 세포의 감염을 도시한다.
도 4는 AAV2 캡시드 단백질 VP1의 아미노산 서열을 제공한다. 아미노산 587 내지 588 (NP)은 굵고 밑줄쳐져 있다.
도 5는 다양한 AAV 혈청형의 AAV 캡시드 단백질 VP1의 아미노산 570-610에 상응하는 아미노산 서열을 제공한다.
도 6A-6C는 AAV 캡시드 단백질 루프 IV (GH 루프) 영역의 아미노산 서열의 정렬을 제공한다. 삽입 부위는 굵은 밑줄로 나타낸다. AAV1: 서열번호:35; AAV6: 서열번호:36; AAV3: 서열번호:37; AAV2: 서열번호:38; AAV8: 서열번호:39; AAV8.1: 서열번호:40; AAV8 rh8: 서열번호:41; AAV10: 서열번호:42; AAV7: 서열번호:43; AAV9: 서열번호:44; AAV 9.1: 서열번호:45; AAV5: 서열번호:46.
도 7A-7V는 예시적인 이종성 유전자 산물의 아미노산 서열을 제공한다.
도 8A-8C는의 아미노산 서열을 제공한다 예시적인 가이드-RNA-지향된 엔도뉴클레아제의 아미노산 서열을 제공한다.
도 2는 녹색 형광 단백질 (GFP)-바코드 작제물을 함유하는 AAV 변이체의 심화 서열분석의 도식적 묘사이다.
도 3은 18-구성원 AAV 변이체 라이브러리에 의해, 신경절 세포 층, 내부 핵 층, 광수용체 층, 및 망막 색소 상피 (RPE) 층에서 세포의 감염을 도시한다.
도 4는 AAV2 캡시드 단백질 VP1의 아미노산 서열을 제공한다. 아미노산 587 내지 588 (NP)은 굵고 밑줄쳐져 있다.
도 5는 다양한 AAV 혈청형의 AAV 캡시드 단백질 VP1의 아미노산 570-610에 상응하는 아미노산 서열을 제공한다.
도 6A-6C는 AAV 캡시드 단백질 루프 IV (GH 루프) 영역의 아미노산 서열의 정렬을 제공한다. 삽입 부위는 굵은 밑줄로 나타낸다. AAV1: 서열번호:35; AAV6: 서열번호:36; AAV3: 서열번호:37; AAV2: 서열번호:38; AAV8: 서열번호:39; AAV8.1: 서열번호:40; AAV8 rh8: 서열번호:41; AAV10: 서열번호:42; AAV7: 서열번호:43; AAV9: 서열번호:44; AAV 9.1: 서열번호:45; AAV5: 서열번호:46.
도 7A-7V는 예시적인 이종성 유전자 산물의 아미노산 서열을 제공한다.
도 8A-8C는의 아미노산 서열을 제공한다 예시적인 가이드-RNA-지향된 엔도뉴클레아제의 아미노산 서열을 제공한다.
정의
용어 "망막 세포"는 본 명세서에서, 망막, 예컨대 망막 신경절 세포; 무축삭 세포; 수평 세포; 두극 세포; 간상 및 원뿔을 포함하는 광수용체 세포; 뮐러 신경교 세포; 별아교세포 (예를 들어, 망막 별아교세포); 및 망막 색소 상피를 포함하는 세포 유형 중 임의의 것을 지칭할 수 있다.
"AAV"는 아데노-관련된 바이러스에 대한 약어이고, 바이러스 자체 또는 이의 유도체를 지칭하기 위해 사용될 수 있다. 용어는 달리 요구되는 경우를 제외하고 모든 하위유형 및 자연 발생 및 재조합 형태 둘 모두를 포함한다. 약어 "rAAV"는 재조합 아데노-관련된 바이러스, 또한 일명 재조합 AAV 벡터 (또는 "rAAV 벡터")을 지칭한다. 용어 "AAV"는 AAV 1형 (AAV-1), AAV 2형 (AAV-2), AAV 유형 3 (AAV-3), AAV 유형 4 (AAV-4), AAV 유형 5 (AAV-5), AAV 유형 6 (AAV-6), AAV 유형 7 (AAV-7), AAV 유형 8 (AAV-8), AAV 유형 9 (AAV-9), 조류 AAV, 소과 AAV, 갯과 AAV, 말 AAV, 영장류 AAV, 비-영장류 AAV, 및 양 AAV을 포함한다. "영장류 AAV"는 영장류로부터 단리된 AAV를 지칭하고, "비-영장류 AAV"는 비-영장류 포유동물 로부터 단리된 AAV르르 지칭하고, "소과 AAV"는 소과 포유동물 (예를 들어, 소), 등 로부터 단리된 AAV를 지칭한다.
본 명세서에서 사용된 바와 같은 "rAAV 벡터"는 AAV 기원이 아닌 폴리뉴클레오타이드 서열 (즉, AAV에 대해 이종성인 폴리뉴클레오타이드), 전형적으로 세포의 유전적 전환을 위한 관심 서열을 포함하는 AAV 벡터를 지칭한다. 일반적으로, 이종성 폴리뉴클레오타이드는 적어도 하나에 의해 측접되고, 그리고 일반적으로 2종의 AAV 역전된 말단 반복 서열 (ITRs)에 의해 측접된다. 용어 rAAV 벡터는 rAAV 벡터 입자 및 rAAV 벡터 플라스미드 둘 모두를 포괄한다.
"AAV 바이러스" 또는 "AAV 바이러스 입자" 또는 "rAAV 벡터 입자"는 (전형적으로 야생형 AAV의 모든 캡시드 단백질에 의한) 적어도 하나의 AAV 캡시드 단백질 및 캡시드화된 폴리뉴클레오타이드 rAAV 벡터로 구성된 바이러스 입자를 지칭한다. 입자가 이종성 폴리뉴클레오타이드 (즉 야생형 AAV 게놈, 예컨대 포유동물 세포로 전달될 이식유전자 이외의 폴리뉴클레오타이드)를 포함하면, 전형적으로 "rAAV 벡터 입자" 또는 간단히 "rAAV 벡터"로 칭한다. 따라서, rAAV 입자의 생산은 반드시 rAAV 벡터의 생산을 포함하는데, 그와 같은 벡터는 rAAV 입자 내에 함유되기 때문이다.
"패키징"은 AAV 입자의 어셈블리 및 캡시드화를 초래하는 일련의 세포내 사건을 지칭한다.
AAV "rep" 및 "cap" 유전자는 아데노-관련된 바이러스의 복제 및 캡시드화 단백질을 인코딩하는 폴리뉴클레오타이드 서열을 지칭한다. AAV rep 및 cap은 본 명세서에서 일명 AAV "패키징 유전자"이다.
AAV에 대한 "헬퍼 바이러스"는, AAV (예를 들어 야생형 AAV)가 포유동물 세포에 의해 복제 및 포장될 수 있는 바이러스를 지칭한다. AAV에 대한 다양한 그와 같은 헬퍼 바이러스는 당해 기술에 공지되어 있고, 아데노바이러스, 헤르페스바이러스 및 폭스바이러스 예컨대 백시니아를 포함한다. 아데노바이러스는 수많은 상이한 하위그룹을 포괄하지만, 하위그룹 C의 아데노바이러스 유형 5는 가장 통상적으로 사용된다. 인간, 비-인간 포유동물 및 조류 기원의 수많은 아데노바이러스는 공지되어 있고 보관소 예컨대 ATCC로부터 이용가능하다. 헤르페스 계열의 바이러스는, 예를 들어, 단순 포진 바이러스 (HSV) 및 엡슈타인-바르 바이러스 (EBV), 뿐만 아니라 사이토메갈로바이러스 (CMV) 및 가성광견병 바이러스 (PRV)을 포함하는데; 이들은 또한 보관소 예컨대 ATCC로부터 이용가능하다.
"헬퍼 바이러스 기능(들)"는 (본 명세서에 기재된 복제 및 패키징에 대한 다른 요건과 관련하여) AAV 복제 및 패키징을 허용하는 헬퍼 바이러스 게놈 에서 인코딩된 기능(들)을 지칭한다. 본 명세서에서 기재된 바와 같이, "헬퍼 바이러스 기능"은 헬퍼 바이러스를 제공하거나 또는, 예를 들어, 트랜스에서 생산제 세포에 대한 필수 기능(들)을 인코딩하는 폴리뉴클레오타이드 서열을 제공하는 것을 포함하여 수 많은 방법으롤 제공될 수 있다.
"감염성" 바이러스 또는 바이러스 입자는, 바이러스성 종이 굴성인 세포에 전달할 수 있는 폴리뉴클레오타이드 성분을 포함하는 것이다. 상기 용어가 바이러스의 임의의 복제 수용력을 반드시 암시하는 것은 아니다. 본 명세서에서 사용된 바와 같이, "감염성" 바이러스 또는 바이러스 입자는 표적 세포에 접근하고, 표적 세포를 감염시키고 그리고 표적 세포에서 이종성 핵산을 발현시킬 수 있는 것이다. 따라서, "감염성"는 표적 세포에 접근하고, 표적 세포를 감염시키고 그리고 표적 세포에서 이종성 핵산을 발현시키기 위한 바이러스 입자의 능력을 지칭한다. 감염성은 시험관내 감염성 또는 생체내 감염성을 지칭할 수 있다. 감염성 바이러스성 입자를 계수하기 위한 검정은 본 개시내용의 다른 곳에 그리고 기술 분야에 기재되어 있다. 바이러스성 감염성은 감염성 바이러스성 입자 대 총 바이러스성 입자의 비로서 표현될 수 있다. 총 바이러스성 입자는 바이러스 게놈 (vg) 복제물의 수로 표현될 수 있다. 세포에서 이종성 핵산을 발현시키기 위한 바이러스 입자의 능력은 "형질도입"으로 칭할 수 있다. 세포에서 이종성 핵산을 발현시키기 위한 바이러스 입자의 능력은 마커 유전자의 평가, 예컨대 녹색 형광 단백질 (GFP) 검정 (예를 들어, 상기 바이러스는 뉴클레오타이드 서열 인코딩 GFP을 포함하는, 경우, GFP는 바이러스 입자로 감염된 세포에서 생산되고 검출되고/거나 측정되는 경우; 또는 예를 들어 효소-결합 면역흡착 검정 (ELISA)에 의해 생상된 단백질의 측정으르 포함하는 수많은 기술을 사용하여 분석될 수 있다. 바이러스성 감염성은 감염성 바이러스성 입자 대 총 바이러스성 입자의 비로서 표현될 수 있다. 감염성 바이러스 입자 대 총 바이러스 입자의 비를 결정하는 방법은 당해 기술에 공지되어 있다. 참고, 예를 들어, Grainger 등 (2005) Mol. Ther. 11:S337 (TCID50 감염성 역가 검정을 기재함); 및 Zolotukhin 등 (1999) Gene Ther. 6:973.
"복제 가능" 바이러스 (예를 들어 복제 가능 AAV)는 감염성이고, (즉 헬퍼 바이러스 또는 헬퍼 바이러스 기능의 존재에서) 감염된 세포에서 복제될 수 있는 표현형 야생형 바이러스를 지칭한다. AAV의 경우에, 복제 능숙도는 일반적으로 기능적 AAV 패키징 유전자의 존재를 필요로 한다. 일반적으로, 본 명세서에서 기재된 바와 같은 rAAV 벡터는 하나 이상의 AAV 패키징 유전자의 결여 때문에 포유동물 세포에서 (특히 인간 세포에서) 복제 불능이다. 전형적으로, 그와 같은 rAAV 벡터는, 복제 가능 AAV이 AAV 패키징 유전자와 유입 rAAV 벡터 사이의 재조합에 의해 생성된 가능성을 최소화하기 위해 임의의 AAV 패키징 유전자 서열이 결여되어 있다. 많은 구현예에서, 본 명세서에서 기재된 바와 같은 rAAV 벡터 제조는, 있다면, 임의의 복제 가능 AAV를 거의 함유하지 않는 것들이다 (rcAAV, 또한 일명 RCA) (예를 들어, 약 1 rcAAV/102 rAAV 미만의 입자, 약 1 rcAAV/104 rAAV 미만의 입자, 약 1 rcAAV/108 rAAV 미만의 입자, 약 1 rcAAV/1012 rAAV 미만의 입자, 또는 rcAAV 없음).
용어 "폴리뉴클레오타이드"는 데옥시리보뉴클레오타이드 또는 리보뉴클레오타이드, 또는 그것의 유사체를 포함하는, 임의의 길이의 뉴클레오타이드의 중합체 형태를 지칭한다. 폴리뉴클레오타이드는 변형된 뉴클레오타이드, 예컨대 메틸화된 뉴클레오타이드 및 뉴클레오타이드 유사체를 포함할 수 있고, 비-뉴클레오타이드 성분에 의해 차단될 수 있다. 존재한다면, 뉴클레오타이드 구조에 대한 변경은 중합체의 어셈블리 전 또는 후에 부여될 수 있다. 본 명세서에서 사용된 바와 같은 용어 폴리뉴클레오타이드는, 상호교환적으로 이중- 및 단일가닥 분자를 지칭한다. 달리 구체화되지 않거나 요구되지 않는 한, 폴리뉴클레오타이드인 본 명세서에 기재된 본 발명의 임의의 구현예는 이중-가닥 형태를 구성하는 것으로 공지되거나 예상된 이중-가닥 형태 및 각각의 2종의 상보적 단일가닥 형태 둘 모두를 포괄한다.
폴리뉴클레오타이드 또는 폴리펩타이드는, 정렬된 경우, 염기 또는 아미노산의 백분율이 2개의 서열을 비교하는 경우, 동일하다는 것을 의미하는, 또 다른 폴리뉴클레오타이드 또는 폴리펩타이드에 대해 특정 비율의 "서열 동일성"을 갖는다. 서열 유사성은 여러 가지 다른 방식으로 결정될 수 있다. 서열 동일성을 결정하기 위해, 서열은, ncbi.nlm.nih.gov/BLAST/에서 월드와이드웹으로 이용가능한, BLAST를 포함하는, 방법 및 컴퓨터 프로그램을 사용하여 정렬될 수 있다. 또 다른 정렬 알고리즘은, Oxford Molecular Group, Inc.의 전체 지분 보유 자회사인, Madison, Wisconsin, USA로부터, Genetics Computing Group (GCG) 패키지에서 이용가능한, FASTA이다. 다른 정렬 기술은 하기에서 기재된다: Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. 특히 흥미로운 것은 서열에서 갭을 허용하는 정렬 프로그램이다. Smith-Waterman은 서열 정렬에서 갭을 허용하는 알고리즘의 한 유형이다. 참조 Meth. Mol. Biol. 70: 173-187 (1997). 또한, Needleman 및 Wunsch 정렬 방법을 사용하는 GAP 프로그램은 서열을 정렬하는데 이용될 수 있다. 참조 J. Mol. Biol. 48: 443-453 (1970)
관심 대상은 서열 동일성을 얻기 위해 Smith Waterman (Advances in Applied Mathematics 2: 482-489 (1981)의 국부 상동성 알고리즘 을 사용하는 BestFit 프로그램이다. 갭 생성 페널티는 일반적으로 1 내지 5, 일반적으로 2 내지 4의 범위일 것이고, 많은 구현예에서 3일 것이다. 갭 연장 패널티는 일반적으로 약 0.01 내지 0.20의 범위일 것이고, 많은 사례에서 0.10일 것이다. 프로그램은 비교하기 위해 입력된 서열에 의해 결정된 디폴트 파라미터를 갖는다. 바람직하게는, 서열 동일성은 프로그램에 의해 결정된 디폴트 파라미터를 사용하여 결정된다. 이러한 프로그램은 또한 Genetics Computing Group (GCG) 패키지 (Madison, Wisconsin, USA)로부터 이용가능하다.
관심 또 다른 프로그램은 FastDB 알고리즘이다. FastDB는 하기에 기재된다: Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. 퍼센트 서열 동일성은 하기 파라미터를 기반으로 하는 FastDB에 의해 계산된다:
미스매치 페널티:
1.00;
갭 페널티:
1.00;
갭 크기 페널티:
0.33; 및
결합 페널티:
30.0.
"유전자"는 전사되고 번역된 후 특정 단백질을 인코딩할 수 있는 적어도 하나의 열린 해독틀 을 함유하는 폴리뉴클레오타이드를 지칭한다.
용어 "가이드 RNA"는, 본 명세서에서 사용된 바와 같이, 하기를 포함하는 RNA를 지칭한다: i) 가이드 RNA-지향된 엔도뉴클레아제 (예를 들어, 부류 2 CRISPR/Cas 엔도뉴클레아제 예컨대 유형 II, 유형 V, 또는 유형 VI CRISPR/Cas 엔도뉴클레아제)에 결합하고 RNA-지향된 엔도뉴클레아제를 활성화시키는 "활성제" 뉴클레오타이드 서열; 및 ii) 표적 핵산과 혼성화되는 뉴클레오타이드 서열을 포함하는 "타겟터" 뉴클레오타이드 서열. "활성제" 뉴클레오타이드 서열 및 "타겟터" 뉴클레오타이드 서열은 별개의 RNA 분자 (예를 들어, "이중-가이드 RNA") 상에 있을 수 있거나; 또는 동일한 RNA 분자 ("단일-가이드 RNA") 상에 있을 수 있다.
"작은 간섭하는" 또는 "짧은 간섭 RNA" 또는 siRNA는 관심 유전자 ("표적 유전자")에 표적화된 뉴클레오타이드의 RNA 듀플렉스이다. "RNA 듀플렉스"는 RNA 분자의 2개의 영역 사이의 상보적 짝짓기에 의해 형성된 구조를 지칭한다. siRNA는, siRNA의 듀플렉스 부분의 뉴클레오타이드 서열이 표적화된 유전자의 뉴클레오타이드 서열에 대해 상보적이라는 점에서 유전자에 대해 "표적화된다". 일부 구현예에서, siRNA의 듀플렉스의 길이는 30 뉴클레오타이드 미만이다. 일부 구현예에서, 듀플렉스는 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 또는 10 개의 뉴클레오타이드일 수 있다. 일부 구현예에서, 듀플렉스의 길이는 19-25 개의 뉴클레오타이드이다. RNA siRNA의 듀플렉스 부분은 헤어핀 구조의 일부일 수 있다. 듀플렉스 부분 외에, 헤어핀 구조는 듀플렉스를 형성하는 2개의 서열 사이에 배치된 루프부를 함유할 수 있다. 루프는 그 길이가 변할 수 있다. 일부 구현예에서 루프는 5, 6, 7, 8, 9, 10, 11, 12 또는 13 개의 뉴클레오타이드. 헤어핀 구조는 또한 3' 또는 5' 돌출부를 함유할 수 있다. 일부 구현예에서, 돌출부는 0, 1, 2, 3, 4 또는 5 개의 뉴클레오타이드의3' 또는 5' 돌출부이다.
본 명세서에서 사용된 바와 같이, 용어 "microRNA"는 내인성 microRNA 및 인공 microRNA (예를 들어, 합성 miRNA)를 비제한적으로 포함하는 임의의 유형의 간섭 RNA를 지칭한다. 내인성 microRNA은 mRNA의 생산적 이용을 조절할 수 있는 게놈에서 자연 인코딩된 작은 RNA 이다. 인공 microRNA는 mRNA의 활성을 조절할 수 있는, 내인성 microRNA 이외의 임의의 유형의 RNA 서열 일 수 있다. microRNA 서열은 이들 서열 중 하나 이상으로 구성된 RNA 분자일 수 있다. MicroRNA (또는 "miRNA") 서열은 하기의 예의 공보에서 기재되었다: Lim, 등, 2003, Genes & Development, 17, 991-1008, Lim 등, 2003, Science, 299, 1540, Lee 및 Ambrose, 2001, Science, 294, 862, Lau 등, 2001, Science 294, 858-861, Lagos-Quintana 등, 2002, Current Biology, 12, 735-739, Lagos-Quintana 등, 2001, Science, 294, 853-857, 및 Lagos-Quintana 등, 2003, RNA, 9, 175-179. microRNA의 예는 더 큰 RNA의 단편이거나 miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, 또는 다른 작은 비-코딩 RNA인 임의의 RNA을 포함한다. 참고, 예를 들어, US 특허 출원 20050272923, 20050266552, 20050142581, 및 20050075492. "microRNA 전구체" (또는 "pre-miRNA")는 본 명세서에 편입된 microRNA 서열을 갖는 스템-루프 구조를 갖는 핵산을 지칭한다. "성숙한 microRNA" (또는 "성숙한 miRNA")는 microRNA 전구체 ("사전-miRNA")로부터 절단되었거나, 또는 합성되었고 (예를 들어, 무세포 합성에 의해 실험실에서 합성되었고), 그리고 약 19 뉴클레오타이드 내지 약 27 뉴클레오타이드의 길이를 갖고, 예를 들어, 성숙한 microRNA는 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 또는 27 nt의 길이를 가질 수 있는 microRNA을 포함한다. 성숙한 microRNA는 표적 mRNA에 결합하고 상기 표적 mRNA의 번역을 억제할 수 있다.
폴리뉴클레오타이드에 적용되는 "재조합"은, 폴리뉴클레오타이드가 클로닝, 제한 또는 결찰 단계, 및 자연에서 발견되는 폴리뉴클레오타이드로부터 구별되는 작제물을 생성하는 다른 절차의 다양한 조합의 산물임을 의미한다. 재조합 바이러스는 재조합 폴리뉴클레오타이드를 포함하는 바이러스 입자이다. 용어들 각각은 최초 폴리뉴클레오타이드 복제물 및 최초 바이러스 작제물의 자손을 포함한다.
"조절 요소" 또는 "조절 서열"은 폴리뉴클레오타이드의 복제, 중복, 전사, 스플라이싱, 번역, 또는 열화를 포함하는 폴리뉴클레오타이드의 기능적 조절에 기여하는 분자의 상호작용과 관련된 뉴클레오타이드 서열이다. 조절은 과정의 빈도, 속도, 또는 특이성에 영향을 줄 수 있고, 본질적으로 증진 또는 억제성일 수 있다. 기술 분야에서 공지된 조절 요소는, 예를 들어, 전사 조절 서열 예컨대 프로모터 및 증진제를 포함한다. 프로모터는 특정 조건 하에서 RNA 중합효소에 결합할 수 있고 위치한 프로모터로부터 다운스트림으로 (3' 방향으로) 일반적으로 위치한 코딩 영역의 전사를 개시할 수 있는 DNA 영역이다.
"작동가능하게 연결된" 또는 "작동적으로 연결된"은 유전 요소의 병치를 언급하고, 여기서 상기 요소는 예상된 방식으로 이들을 작동시키는 관계에 있다. 예를 들면, 프로모터가 코딩 서열의 전사 개시를 도우면 프로모터는 코딩 영역에 작동가능하게 연결된다. 이러한 기능적 관계가 유지되는 한 프로모터와 코딩 영역 사이 개입하는 잔기가 있을 수 있다.
"발현 벡터"는 관심의 폴리펩타이드를 인코딩하는 영역을 구성하는 벡터이고, 의도된 표적 세포에서 단백질의 발현에 영향을 미치는 데 사용된다. 발현 벡터는 또한 표적에서 단백질의 발현을 용이하게 하기 위해 인코딩 영역에 작동가능하게 연결된 제어 요소를 포함한다. 제어 요소와 이들이 발현을 위하여 작동가능하게 연결되는 유전자 또는 유전자들과의 조합은 때때로 "발현 카셋트"로서 지칭되고, 다수의 이들은 종래기술에서 공지되고 이용가능하거나 종래기술에서 이용가능한 성분으로부터 용이하게 작제될 수 있다.
"이종성"은 비교되고 있는 나머지 실체의 것으로부터 유전자형질적으로 구별되는 실체로부터 파생된 것을 의미한다. 예를 들어, 유전 공학 기술에 의해 다른 종에서 파생된 플라스미드나 벡터에 도입된 폴리뉴클레오타이드는 이종성 폴리뉴클레오타이드이다. 연결된 채 자연적으로 발견되지 않은 코딩 서열에 작동가능하게 연결된 그리고 이의 자연 코딩 서열에서 제거된 프로모터는 이종성 프로모터이다. 따라서, 예를 들어, 이종성 유전자 생성물을 인코딩하는 이종성 핵산을 포함하는 rAAV는 자연-발생, 야생형 AAV에서 정상적으로 포함되지 않는 핵산을 포함하는 rAAV이고, 인코딩된 이종성 유전자 생성물은 자연-발생, 야생형 AAV에 의해 정상적으로 인코딩되지 않는 유전자 생성물이다. 또 다른 예로서, 캡시드 단백질의 GH 루프에 삽입된 이종성 펩타이드를 포함하는 변이체 AAV 캡시드 단백질은 자연-발생, 야생형 AAV에서 정상적으로 포함되지 않는 펩타이드의 삽입을 포함하는 변이체 AAV 캡시드 단백질이다.
용어 "유전적 변경" 및 "유전적 변형" (및 이의 문법적 변형)은 유전적 요소 (예를 들면, 폴리뉴클레오타이드)가 유사분열 또는 감수분열에 의한 것 이외에 세포에 도입되는 공정을 지칭하기 위해 본 명세서에서 교환가능하게 사용된다. 요소는 세포에 이종성일 수 있거나, 세포에서 이미 존재하는 요소의 추가의 카피 또는 개량된 버전일 수 있다. 유전적 변경은, 예를 들어, 전기천공, 인산칼슘 침전, 또는 폴리뉴클레오타이드-리포솜 복합체와 접촉과 같은, 종래기술에서 공지된 임의의 공정을 통해 재조합 플라스미드 또는 다른 폴리뉴클레오타이드로 세포를 형질감염시킴으로써, 영향받을 수 있다. 유전적 변경은, 예를 들어, DNA 또는 RNA 바이러스 혹은 바이러스 벡터로 형질도입 또는 감염시킴으로써, 또한 영향받을 수 있다. 일반적으로, 유전적 요소는 세포에서 염색체 또는 미니-염색체에 도입되지만; 세포 및 이의 자손의 표현형 및/또는 유전자형을 변화시키는 임의의 변경은 이 용어에 포함된다.
세포는, 서열이 세포 시험관내 세포의 연장된 배양 동안에 그것의 기능 수행하기 위해 이용가능하면 "안정적으로" 변경되고, 형질도입되고, 유전자 변형되거나, 또는 유전적 서열로 전환된 것으로 언급된다. 일반적으로, 그와 같은 세포는, 변화된 세포의 자손에 의해 상속되는 유전적 변이가 도입된다는 점에서 "상속에 의하여 " 변경된다 (유전자 변형된다).
용어들 "폴리펩타이드", "펩타이드", 및 "단백질"은 본 명세서에서 상호교환적으로 사용되고 임의의 길이의 아미노산의 중합체를 지칭한다. 용어들은 또한 변형되었던 아미노산 중합체; 예를 들어, 디설파이드 결합 형성, 당화, 지질화, 인산화, 또는 표지 성분과의 콘주게이션을 포괄한다. 폴리펩타이드 예컨대 항-혈관신생 폴리펩타이드, 신경보호성 폴리펩타이드, 및 등은, 유전자 산물을 포유동물 대상체에게 전달하는 것과 관련하여 논의할 때, 및 그의 조성물은, 온전한 단백질의 원하는 생화학적 기능을 유지하는, 각각의 온전한 폴리펩타이드, 또는 이의 임의의 단편 또는 유전자적으로 조작된 유도체를 지칭한다. 유사하게, 항-혈관신생 폴리펩타이드를 인코딩하는 핵산, 신경보호성 폴리펩타이드를 인코딩하는 핵산, 및 유전자 산물의 포유동물 대상체에의 전달에 사용하기 위한 다른 그와 같은 핵산 (이는 수령체 세포에 전달될 "이식유전자"로 칭할 수 있음)에 대한 언급은, 원하는 생화학적 기능을 보유하고 있는 온전한 폴리펩타이드 또는 임의의 단편 또는 유전자적으로 조작된 유도체를 인코딩하는 폴리뉴클레오타이드를 포함한다.
"단리된" 플라스미드, 핵산, 벡터, 바이러스, 비리온, 숙주 세포, 또는 다른 서브스턴스는 서브스턴스 또는 유사한 서브스턴스가 자연적으로 발생하거나 그로부터 초기에 제조되는 경우 또한 존재할 수 있는 다른 성분의 적어도 일부가 없는 서브스턴스의 제조물을 지칭한다. 따라서, 예를 들어, 단리된 서브스턴스는 공급원 혼합물로부터 그것을 농축시키기 위해 정제 기술을 사용함으로써 제조될 수 있다. 농축은, 절대 기준, 예컨대 용액의 체적당 중량으로 측정될 수 있거나, 공급원 혼합물에서 존재하는 제2, 잠재적으로 방해 서브스턴스에 관련하여 측정될 수 있다. 본 발명의 구현예의 증가하는 농축은 증가적으로 더욱 단리된다. 단리된 플라스미드, 핵산, 벡터, 바이러스, 숙주 세포, 또는 다른 서브스턴스는 일부 구현예에서, 예를 들어, 약 80% 내지 약 90% 순수, 적어도 약 90% 순수, 적어도 약 95% 순수, 적어도 약 98% 순수, 또는 적어도 약 99%, 또는 초과 순수하게 정제된다.
본 명세서에서 사용된 바와 같이, 용어들 "치료", "치료하는", 및 등은, 원하는 약리적 및/또는 생리적 효과를 얻는 것을 지칭한다. 효과는 질환 또는 이의 증상을 완전히 또는 부분적으로 예방하는 관점에서 예방적일 수 있고/거나 질환 및/또는 질환에 대한 부정적 영향에 대한 부분적 또는 완전한 치료의 관점에서 치료적일 수 있다. 본 명세서에서 사용된 바와 같은 "치료"는, 포유동물, 특히 인간에서 질환의 임의의 치료를 포괄하고, 하기를 포함한다: (a) 질환에 취약하거나 질병을 습득할 위험에 있으나, 질환을 갖는 것으로 아직 진단되지 않은 대상체에서 질환이 생기는 것을 예방함; (b) 질환을 억제함, 즉, 그것의 발달의 저지; 및 (c) 질환을 경감시킴, 즉, 질환의 퇴행을 야기함.
용어들 "개체", "숙주", "대상체", 및 "환자"는 본 명세서에서 상호교환적으로 유인원 및 인간; 포유동물 스포츠용 동물 (예를 들어, 말, 낙타, 등); 포유동물 농장 동물 (예를 들어, 양, 염소, 소, 등); 포유동물 애완동물 (개, 고양이, 등); 및 설치류 (예를 들어, 마우스, 랫트, 등)을 포함하는 인간 및 비-인간 영장류를 비제한적으로 포함하는 포유동물을 지칭한다. 일부 경우에, 개체는 인간이다.
본 발명이 추가로 기재되기 전, 본 발명이 기재된 특정한 구현예에 제한되지 않고, 자체로, 물론, 다양할 수 있다는 것이 이해된다. 본 명세서에서 사용된 용어들이 단지 특정한 구현예 기재의 목적을 위한 것이 또한 이해되고, 본 발명의 범위가 첨부된 청구항에 의해서만 제한되지 않을 것이기 때문에, 제한되는 의도는 아니다.
값의 범위가 제공되는 경우, 그 범위 그리고 그 명시된 범위내 임의의 다른 명시된 또는 개재하는 값의 상한과 하한 사이, 문맥이 명확히 달리 나타내지 않는 한 하한의 단위의 10분의 1까지, 각각의 개재하는 값이 본 발명 내에 포괄되는 것이 이해된다. 이들 더 작은 범위의 상한 및 하한은 독립적으로 더 작은 범위에서 포함될 수 있고, 또한 명시된 범위에서 특별히 제외된 한계에 따라 발명에 포괄된다. 명시된 범위가 한쪽 또는 양쪽 한계를 포함하는 경우, 포함된 어느 한쪽 또는 양쪽 한계를 제외한 범위는 또한 본 발명에서 포함된다.
달리 정의되지 않는 한, 본 명세서에서 사용된 모든 기술 및 과학 용어는 본 발명이 속하는 종래기술에서 당업자에 의해 통상적으로 이해되는 바와 동일한 의미를 갖는다. 본 명세서에서 기재된 것과 유사한 또는 등가인 임의의 방법 및 물질이 또한 본 발명의 실시 또는 시험에서 사용될 수 있어도, 바람직한 방법 및 물질은 이제 기재된다. 본 명세서에서 언급된 모든 공보는 그 공보가 인용되는 것과 관련하여 방법 및/또는 물질을 개시 및 기재하기 위해 참조로 본 명세서에서 편입된다.
본 명세서에서 그리고 첨부된 청구항에서 사용된 바와 같이, 단수 형태 "a", "an", 및 "the"는 문맥이 명확하게 달리 나타내지 않는 한 복수의 지시물을 포함하는 것이 주목되어야 한다. 따라서, 예를 들어, "AAV 캡시드" 지칭은 복수의 그와 같은 캡시드를 포함하고 "AAV 비리온" 지칭은 하나 이상의 AAV 비리온 그리고 종래기술의 당업자에 공지된 이의 등가물, 및 기타 등등 지칭을 포함한다. 청구항이 임의의 선택적 요소를 제외하도록 작성될 수 있다는 것이 추가로 주목된다. 이와 같이, 이러한 명시는 청구항 요소의 인용과 관련하여 "단독으로", "단지" 및 기타 등등과 같은 배타적 용어의 사용, 또는 "부정적" 제한의 사용에 대한 선행 기준으로서 제공하기 위한 의도이다.
명확화를 위하여, 개별 구현예의 문맥에서 기재되는, 본 발명의 특정 피쳐가 또한 단일 구현예에서 조합으로 제공될 수 있다는 것이 인정된다. 반대로, 간결함을 위하여, 단일 구현예의 맥락에서 기재되는, 본 발명의 다양한 피쳐가 별도로 또는 임의의 적절한 하위-조합으로 또한 제공될 수 있다. 본 발명에 속하는 구현예의 모든 조합은 본 발명에 의해 구체적으로 포괄되고 각각의 및 모든 조합이 개별적으로 그리고 명시적으로 개시된 것처럼 본 명세서에서 개시된다. 게다가, 이의 다양한 구현예 및 요소의 모든 하위-조합은 또한 본 발명에 의해 구체적으로 포괄되고 각각의 및 모든 이와 같은 하위-조합이 개별적으로 그리고 명시적으로 본 명세서에서 개시된 것처럼 본 명세서에서 개시된다.
본 명세서에서 논의된 공보는 본원의 출원일에 앞서 이들의 개시를 위하여 단독으로 제공된다. 본 발명이 선행 발명의 덕택으로 이와 같은 공보를 전치할 권리가 없다는 것을 인정하는 것으로 해석되는 것이 본 명세서에서 없다. 추가로, 제공된 공보의 일자는 독립적으로 확인될 필요가 있을 수 있는 실제 공보 일자와 상이할 수 있다.
상세한 설명
본 개시내용은 재조합 변경된 캡시드 단백질을 갖는 아데노-관련된 바이러스 (AAV) 비리온을 제공하되, 상기 재조합 AAV (rAAV) 비리온은 야생형 AAV와 비교하여 망막 세포의 더 큰 감염성을 나타내고; 그리고 상기 rAAV 비리온은 이종성 핵산을 포함한다. rAAV 비리온은 망막 세포에 대한 상응하는 야생형 AAV의 감염성과 비교하여 망막 세포의 더 큰 감염성을 나타낸다. 망막 세포는 광수용체 (예를 들어, 간상체; 추상체), 망막 신경절 세포 (RGC), 뮐러 세포 (뮐러 신경교 세포), 별아교세포 (예를 들어, 망막 별아교세포), 두극 세포, 무축삭 세포, 수평 세포, 또는 망막 색소 상피 (RPE) 세포일 수 있다. 본 개시내용은 추가로, 개체에서 유전자 산물을 망막 세포에 전달하는 방법, 및 안구 질환을 치료하는 방법을 제공한다. 본 개시내용은 변경된 캡시드 단백질을 갖는 rAAV 비리온을 제공하되, 상기 rAAV 비리온은 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 또는 망막 색소 상피에 대한 국제화의 정도와 비교하여, 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 및 망막 색소 상피 중 하나 이상에 대해 적어도 5-배수 증가된 국재화를 나타내고; 그리고 상기 rAAV 비리온은 이종성 핵산을 포함한다.
변이체 AAV 캡시드 폴리펩타이드
본 개시내용은 변이체 AAV 캡시드 단백질을 제공한다. 본 개시내용의 변이체 AAV 캡시드 단백질은 친계 AAV 캡시드 단백질의 표면-접근가능한 (예를 들어, 용매-접근가능한) 부분에서의 삽입 부위에서 5개의 아미노산 내지 20개 길이의 아미노산의 이종성 펩타이드의 삽입을 포함하고, 이로써, 상기 변이체 캡시드 단백질은, AAV 비리온 내에 존재할 때, 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 상기 망막 세포의 감염성과 비교하여 망막 세포의 증가된 감염성을 부여한다. 다른 사례에서, 망막 세포는 뮐러 세포. 다른 망막 세포는 무축삭 세포, 두극 세포, 및 수평 세포를 포함한다. "약 5개의 아미노산 내지 약 20개의 아미노산의 삽입"은 또한 본 명세서에서 일명 "펩타이드 삽입" (예를 들어, 이종성 펩타이드 삽입)이다. "상응하는 친계 AAV 캡시드 단백질"은 펩타이드 삽입없는 동일한 AAV 혈청형의 AAV 캡시드 단백질 을 지칭한다. 많은 사례에서, 변이체 AAV 캡시드는 5개의 아미노산 내지 20개 길이의 아미노산 (예를 들어, 5 내지 7, 7 내지 10, 10 내지 12, 12 내지 15, 또는 15 내지 20개 길이의 아미노산)의 단일 이종성 펩타이드 삽입물을 포함한다.
삽입 부위는 AAV 캡시드 단백질의 GH 루프, 또는 루프 IV 내에, 예를 들어, AAV 캡시드 단백질의 GH 루프, 또는 루프 IV의 용매-접근가능한 부분 내에 있다. AAV 캡시드의 GH 루프/루프 IV에 대해, 참고, 예를 들어, van Vliet 등 (2006) Mol. Ther. 14:809; Padron 등 (2005) J. Virol. 79:5047; 및 Shen 등 (2007) Mol. Ther. 15:1955. 예를 들어, 삽입 부위는 도 6A-6C에서 묘사된 바와 같이 AAV 캡시드 단백질의 아미노산 411-650 내에 있을 수 있다. 예를 들어, 삽입 부위는 도 5에서 묘사된 바와 같이, AAV2의 아미노산 570-611 내에, AAV1의 아미노산 571-612 내에, AAV5의 아미노산 560-601 내에, AAV6의 아미노산 571 내지 612 내에, AAV7의 아미노산 572 내지 613 내에, AAV8의 아미노산 573 내지 614 내에, AAV9의 아미노산 571 내지 612 내에, 또는 AAV10의 아미노산 573 내지 614 내에 있을 수 있다. 일부 경우에, 삽입 부위는 AAV2 캡시드 단백질의 아미노산 588 내지 589, 또는 상이한 혈청형의 AAV에서의 상응하는 삽입 부위 사이에 있다. 일부 경우에, 삽입 부위는 AAV2 캡시드 단백질의 아미노산 587 내지 588, 또는 상이한 혈청형의 AAV에서의 상응하는 삽입 부위 사이에 있다.
일부 경우에, 약 5개의 아미노산 내지 약 20개 길이의 아미노산 (예를 들어, 5 내지 7, 7 내지 10, 10 내지 12, 12 내지 15, 또는 15 내지 20개 길이의 아미노산)의 이종성 펩타이드는 상응하는 친계 AAV 캡시드 단백질에 관한 캡시드 단백질의 GH 루프 또는 루프 IV에서의 삽입 부위 내에 삽입된다. 예를 들어, 삽입 부위는 AAV2의 아미노산 587 내지 588, 또는 또 다른 AAV 혈청형의 캡시드 하위단위의 상응하는 위치일 수 있다. 삽입 부위 587/588이 AAV2 캡시드 단백질을 기반으로 함을 유의해야 한다. 5개의 아미노산 내지 약 20개 길이의 아미노산 (예를 들어, 5 내지 7, 7 내지 10, 10 내지 12, 12 내지 15, 또는 15 내지 20개 길이의 아미노산)의 이종성 펩타이드는 AAV2 (예를 들어, AAV8, AAV9, 등) 이외의 AAV 혈청형에서의 상응하는 부위 내에 삽입될 수 있다. 당해 분야의 숙련가는 다양한 AAV 혈청형의 캡시드 단백질의 아미노산 서열의 비교를 기반으로 공지되어 있고, "AAV2의 아미노산 587-588에 상응하는" 삽입 부위는 임의의 주어진 AAV 혈청형의 캡시드 단백질 내에 있다. 다양한 AAV 혈청형의 캡시드 단백질 AAV2의 VP1의 아미노산 570-611에 상응하는 서열 (참조 도 4)은 도 5에 도시되어 있다. 참고, 예를 들어, 유전자은행 수탁 번호 NP_049542 for AAV1; 유전자은행 수탁 번호 AAD13756 for AAV5; 유전자은행 수탁 번호 AAB95459 for AAV6; 유전자은행 수탁 번호 YP_077178 for AAV7; 유전자은행 수탁 번호 YP_077180 for AAV8; 유전자은행 수탁 번호 AAS99264 for AAV9 및 유전자은행 수탁 번호 AAT46337 for AAV10.
예를 들어, 삽입 부위는 AAV2의 아미노산 587 내지 588, AAV1의 아미노산 590 내지 591, AAV5의 아미노산 575 내지 576, AAV6의 아미노산 590 내지 591, AAV7의 아미노산 589 내지 590, AAV8의 아미노산 590 내지 591, AAV9의 아미노산 588 내지 589, 또는 AAV10의 아미노산 588 내지 589일 수 있다. 삽입 부위는 도 5에서 밑줄쳐져 있고; 상기 아미노산 넘버링은 도 5에서 묘사된 넘버링을 기반으로 한다.
일부 구현예에서, 당해 캡시드 단백질은 도 6A-6C에서 제시된 아미노산 서열 에 대해 적어도 약 85%, 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하고; 그리고 5 내지 20개 길이의 아미노산 (예를 들어, 5 내지 7, 7 내지 10, 10 내지 12, 12 내지 15, 또는 15 내지 20개 길이의 아미노산)의 이종성 펩타이드의 삽입을 갖는 GH 루프를 포함한다.
삽입 펩타이드
전술한 바와 같이, 길이가 약 5개의 아미노산 내지 약 20개의 아미노산인 이종성 펩타이드는 AAV 캡시드의 GH 루프에 삽입된다. 일부 경우에, 삽입 펩타이드는 5개의 아미노산 내지 20개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 7개의 아미노산 내지 15개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 9개의 아미노산 내지 15개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 9개의 아미노산 내지 12개의 아미노산. 삽입 펩타이드는 5개의 아미노산, 6개의 아미노산, 7개의 아미노산, 8개의 아미노산, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 12개의 아미노산, 13개의 아미노산, 14개의 아미노산, 15개의 아미노산, 16개의 아미노산, 17개의 아미노산, 18개의 아미노산, 19개의 아미노산, 또는 20개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 7개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 8개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 9개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 10개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 11개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 12개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 13개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 14개의 아미노산의 길이를 갖는다. 일부 경우에, 삽입 펩타이드는 15개의 아미노산의 길이를 갖는다.
펩타이드 삽입물은, 일부 경우에, 식 I의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu, Ile, Pro, 또는 Gln이고;
X2는 Ala, Pro, Ser, Asp, Gly, Thr, 또는 Val이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, 또는 Tyr이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, 또는 Arg이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, 또는 Tyr이고;
X6는 Thr, Ala, Gln, Ser, Glu, Pro, 또는 Ile;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, 또는 Cys이고;
X8는 Lys, Ser, Arg, Thr, Ala, Glu, Ile, 또는 Asn이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Phe, Asp, Thr, Val, 또는 Met이다.
식 I의 펩타이드 삽입물은, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (23) LAKADETRPA (서열번호:69); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); (32) LAISDQSKPA (서열번호:78); (33) LADATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (35) LAKSDQSRPA (서열번호:81); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (39) LPISDQTKHA (서열번호:85); (40) LPKDATKTIA (서열번호:86); (41) LPPQDTTKNA (서열번호:87); (42) PAPQDTTKNA (서열번호:88); (43) QAHQDTTKNA (서열번호:89); (44) LAHETSPRPA (서열번호:90); (45) LAKSTSTAPA (서열번호:91); (46) LADQDTTKNA (서열번호:92); (47) LAESDQSKPA (서열번호:93); (48) LAHKDTTKNA (서열번호:94); (49) LAHKTQQKM (서열번호:95); (50) LAHQDTTENA (서열번호:96); (51) LAHQDTTINA (서열번호:97); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); (56) LAHQDTTKTM (서열번호:102); (57) LAHQNTTKNA (서열번호:103); (58) LAHRDTTKNA (서열번호:104); (59) LAISDQTNHA (서열번호:105); (60) LAKQKSASTA (서열번호:106); (61) LAKSDQCKPA (서열번호:107); (62) LAKSDQSKPD (서열번호:108); (63) LAKSDQSNPA(서열번호:109); (64) LAKSYQSKPA (서열번호:110); (65) LANQDTTKNA (서열번호:111); (66) LAPQNTTKNA (서열번호:112); (67) LAPSSIQKPA (서열번호:113); (68) LAQQDTTKNA (서열번호:114); (69) LAYQDTTKNA (서열번호:115); (70) LDHQDTTKNA (서열번호:116); (71) LDHQDTTKSA (서열번호:117); (72) LGHQDTTKNA (서열번호:118); (73) LPHQDTTKND (서열번호:119); (74) LPHQDTTKNT (서열번호:120); (75) LPHQDTTNNA (서열번호:121); (76) LTHQDTTKNA (서열번호:122); (77) LTKDATKTIA (서열번호:123); (78) LTPQDTTKNA (서열번호:124); 및 (79) LVHQDTTKNA (서열번호:125).
펩타이드 삽입물은, 일부 경우에, 식 II의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala이다.
식 II의 펩타이드 삽입물은, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA(서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (23) LAKADETRPA (서열번호:69); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); (32) LAISDQSKPA (서열번호:78); (33) LADATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (35) LAKSDQSRPA (서열번호:81); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (39) LPISDQTKHA (서열번호:85); (40) LPKDATKTIA (서열번호:86); (41) LPPQDTTKNA (서열번호:87); 및 (42) PAPQDTTKNA (서열번호:88).
식 II의 펩타이드는, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); 및 (18) LAVSDSTKAA (서열번호:64). 일부 경우에, 펩타이드 삽입물은 (1) LAKDATKNA (서열번호:47)이다. 일부 경우에, 펩타이드 삽입물은 (2) PAHQDTTKNA (서열번호:48)이다. 일부 경우에, 펩타이드 삽입물은 (3) LAHQDTTKNA (서열번호:49)이다. 일부 경우에, 펩타이드 삽입물은 (4) LATTSQNKPA (서열번호:50)이다. 일부 경우에, 펩타이드 삽입물은 (5) LAISDQTKHA (서열번호:51)이다. 일부 경우에, 펩타이드 삽입물은 (6) IARGVAPSSA (서열번호:52)이다. 일부 경우에, 펩타이드 삽입물은 (7) LAPDSTTRSA (서열번호:53)이다. 일부 경우에, 펩타이드 삽입물은 (8) LAKGTELKPA (서열번호:54)이다. 일부 경우에, 펩타이드 삽입물은 (9) LAIIDATKNA (서열번호:55)이다. 일부 경우에, 펩타이드 삽입물은 (10) LAVDGAQRSA (서열번호:56)이다. 일부 경우에, 펩타이드 삽입물은 (11) PAPQDTTKKA (서열번호:57)이다. 일부 경우에, 펩타이드 삽입물은 (12) LPHQDTTKNA (서열번호:58)이다. 일부 경우에, 펩타이드 삽입물은 (13) LAKDATKTIA (서열번호:59)이다. 일부 경우에, 펩타이드 삽입물은 (14) LAKQQSASTA (서열번호:60)이다. 일부 경우에, 펩타이드 삽입물은 (15) LAKSDQSKPA (서열번호:61)이다. 일부 경우에, 펩타이드 삽입물은 (16) LSHQDTTKNA (서열번호:62)이다. 일부 경우에, 펩타이드 삽입물은 (17) LAANQPSKPA (서열번호:63)이다. 일부 경우에, 펩타이드 삽입물은 (18) LAVSDSTKAA (서열번호:64)이다.
펩타이드 삽입물은, 일부 경우에, 식 III의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Thr, Asp Val, 또는 Met이다.
식 III의 펩타이드 삽입물은, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (6) IARGVAPSSA (서열번호:52);(7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60);(16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (31) LAHQVTTKNA (서열번호:77); (33) LAKDATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (41) LPPQDTTKNA (서열번호:87); (42) PAPQDTTKNA (서열번호:88); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); (56) LAHQDTTKTM (서열번호:102); (73) LPHQDTTKND (서열번호:119); 및 (74) LPHQDTTKNT (서열번호:120).
펩타이드 삽입물은, 일부 경우에, 식 IV의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu이고;
X2는 Ala이고;
X3는 Lys, His, Thr, Ile, Pro, 또는 Val이고;
X4 (존재하면)는 Gln, Asp, Ser, 또는 Gly이고;
X5는 Asp, Ser, 또는 Gln이고;
X6는 Thr, Ala, Gln, 또는 Ser이고;
X7는 Thr 또는 Ser이고;
X8는 Lys, Ser, 또는 Arg이고;
X9는 Asn, Pro, 또는 Ser이고; 그리고
X10는 Ala이다.
식 IV의 펩타이드 삽입물은, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (3) LAHQDTTKNA (서열번호:49); (7) LAPDSTTRSA (서열번호:53); (15) LAKSDQSKPA (서열번호:61); (20) LAPDQTTRNA (서열번호:66); (22) LAPQDTTKNA (서열번호:68) ;(28) LAHQDTTRNA (서열번호:74); (32) LAISDQSKPA (서열번호:78); (34) LAKDTTKNA (서열번호:80); 및 (35) LAKSDQSRPA (서열번호:81).
펩타이드 삽입물은, 일부 경우에, 식 V의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu이고;
X2는 Ala이고;
X3는 Lys 또는 His이고;
X4 (존재하면)는 Gln, Asp, Ser, 또는 Gly이고;
X5는 Asp, Ser, 또는 Gln이고;
X6는 Thr, Ala, Gln, 또는 Ser이고;
X7는 Thr 또는 Ser이고;
X8는 Lys, Ser, 또는 Arg이고;
X9는 Asn, Pro, 또는 Ser이고; 그리고
X10는 Ala이다.
식 V의 펩타이드 삽입물은, 비제한적으로 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (15) LAKSDQSKPA (서열번호:51); (34) LAKDTTKNA (서열번호:80); 및 (35) LAKSDQSRPA (서열번호:81).
펩타이드 삽입물은, 일부 경우에, 식 VI의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중,
X1는 Leu이고;
X2는 Ala이고;
X3는 Asn, Lys, Thr, Gln, Ser, Ile, 또는 Leu이고;
X4는 Ser, Ala, Thr, Glu, Gln, Gly, Lys, 또는 Pro이고;
X5는 Asp, Pro, Glu, Thr, Asn, 또는 Arg이고;
X6는 Ile, His, Thr, Gln, Asn, Tyr, Asp, 또는 Glu이고;
X7는 Gln, Thr, Asn, Ala, 또는 Lys이고;
X8는 Lys, Thr, Arg, 또는 Asp이고;
X9는 Pro, Asn, Thr, Arg, Lys, 또는 Ser이고; 그리고
X10는 Ala이다.
식 VI의 펩타이드는, 비제한적으로 하기를 포함한다: (80) LAKANQNTPA (서열번호:126); (81) LATTPITKPA (서열번호:127); (82) LATTPIAKPA (서열번호:128); (83) LAIEDHTKSA (서열번호:129); (84) LAQSEHQRPA (서열번호:130); (85) LAKSPNKDNA (서열번호:131); (86) LANQDYTKTA (서열번호:132); (87) LANSTDQTRA (서열번호:133); (88) LALGETTRPA (서열번호:134); (89) LANSTEQTRA (서열번호:135); (90) LAQADTTKNA (서열번호:136); (91) LASKDITKTA (서열번호:137); 및 (92) LASPRHNKKC (서열번호:138).
일부 경우에, 펩타이드 삽입물은 식 VII: LAHQDTTKX1X2X3 (서열번호:148)의 펩타이드이되, 상기 X1는 Lys, Thr, Asn, 또는 His이고; X2는 Ala, Thr, Val, Ile, Met, 또는 Asp이고; 그리고 X3는, 존재한다면, Ala이다. 식 VII의 펩타이드는, 비제한적으로 하기를 포함한다: (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); (56) LAHQDTTKTM (서열번호:102); 및 (93) LAHQDTTKTIA (서열번호:139).
일부 경우에, 펩타이드 삽입물은 식 VIII: LAX1QX2TX3X4X5X6 (서열번호:149)의 펩타이드이되, 상기 X1는 Ala, Pro, Asp, 또는 His이고; X2는 Gly 또는 Asp이고; X3는 Ala, Thr, 또는 Lys이고; X4는 Asn, Glu, Lys, Arg, 또는 Thr이고; X5는 Leu, Asn, Lys, 또는 Thr이고; 그리고 X6는, 존재한다면, Ala, Thr, Asp, Val, 또는 Met이다. 식 VIII는의 펩타이드는, 비제한적으로 하기를 포함한다: (94) LAAQGTANL (서열번호:140); (22) LAPQDTTKNA (서열번호:68); (46) LADQDTTKNA (서열번호:92); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTTNA (서열번호:75); (50) LAHQDTTENA (서열번호:96); (51) LAHQDTTINA (서열번호:97); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); 및 (56) LAHQDTTKTM (서열번호:102).
일부 경우에, 펩타이드 삽입물은 식 IX: X1AX2X3DX4TKX5A (서열번호:150)의 펩타이드이되, 상기 X1는 Val 또는 Leu이고; X2는 Ile, Val, His, 또는 Asp이고; X3는 Glu, Ser, Lys, 또는 Gln이고; X4는 His, Ser, 또는 Thr이고; 그리고 X5는 Ser, Ala, Asn, His, 또는 Lys이다. 식 IX의 펩타이드는, 비제한적으로 하기를 포함한다: (95) VAIEDHTKSA (서열번호:141); (18) LAVSDSTKAA (서열번호:64); (46) LADQDTTKNA (서열번호:92); (48) LAHKDTTKNA (서열번호:94); (26) LAHQDTTKHA (서열번호:72); 및 (27) LAHQDTTKKA (서열번호:73).
일부 경우에, 펩타이드 삽입물은 식 X: X1X2X3AX4QX5TX6KNA (서열번호:151)의 펩타이드이되, 상기 X1는, 존재한다면, Leu이고; X2는, 존재한다면, Ala이고; X3는 Lys, Leu, 또는 Pro이고; X4는 Asn, His, Pro, 또는 Tyr이고; X5는 Asn, Gly, Val, 또는 Asp이고; 그리고 X6는 Pro 또는 Thr이다. 식 X의 펩타이드는, 비제한적으로 하기를 포함한다: (96) LAKANQNTPKNA (서열번호:142); (57) LAHQNTTKNA (서열번호:103); (66) LAPQNTTKNA (서열번호:112); (69) LAYQDTTKNA (서열번호:115); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); 및 (42) PAPQDTTKNA (서열번호:88).
일부 경우에, 펩타이드 삽입물은 LAHQDTTKKX (서열번호:143)이되, 상기 X는 임의의 아미노산. 일부 경우에, 펩타이드 삽입물은 LAHQDTTKKX (서열번호:143)이되, 상기 X는 Ala, Thr, Asp, Val, 또는 Met이다. 일부 경우에, 펩타이드 삽입물은 (27) LAHQDTTKKA (서열번호:73)이다. 일부 경우에, 펩타이드 삽입물은 (52) LAHQDTTKKT (서열번호:98)이다. 일부 경우에, 펩타이드 삽입물은 LAHQDTTKKD (서열번호:144)이다. 일부 경우에, 펩타이드 삽입물은 LAHQDTTKKV (서열번호:145)이다. 일부 경우에, 펩타이드 삽입물은 LAHQDTTKKM (서열번호:146)이다.
일부 경우에, 펩타이드 삽입물은 (88) LALGETTRPA (서열번호:134)가 아니다. 일부 경우에, 펩타이드 삽입물은 LGETTRP (서열번호:147)가 아니다.
적합한 펩타이드 삽입물은, 비제한적으로, 하기를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (23) LAKADETRPA (서열번호:69); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); (32) LAISDQSKPA (서열번호:78); (33) LADATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (35) LAKSDQSRPA (서열번호:81); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (39) LPISDQTKHA (서열번호:85); (40) LPKDATKTIA (서열번호:86); (41) LPPQDTTKNA (서열번호:87); (42) PAPQDTTKNA (서열번호:88); (43) QAHQDTTKNA (서열번호:89); (44) LAHETSPRPA (서열번호:90); (45) LAKSTSTAPA (서열번호:91); (46) LADQDTTKNA (서열번호:92); (47) LAESDQSKPA (서열번호:93); (48) LAHKDTTKNA (서열번호:94); (49) LAHKTQQKM (서열번호:95); (50) LAHQDTTENA (서열번호:96); (51) LAHQDTTINA (서열번호:97); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); (56) LAHQDTTKTM (서열번호:102); (57) LAHQNTTKNA (서열번호:103); (58) LAHRDTTKNA (서열번호:104); (59) LAISDQTNHA (서열번호:105); (60) LAKQKSASTA (서열번호:106); (61) LAKSDQCKPA (서열번호:107); (62) LAKSDQSKPD (서열번호:108); (63) LAKSDQSNPA(서열번호:109); (64) LAKSYQSKPA (서열번호:110); (65) LANQDTTKNA (서열번호:111); (66) LAPQNTTKNA (서열번호:112); (67) LAPSSIQKPA (서열번호:113); (68) LAQQDTTKNA (서열번호:114); (69) LAYQDTTKNA (서열번호:115); (70) LDHQDTTKNA (서열번호:116); (71) LDHQDTTKSA (서열번호:117); (72) LGHQDTTKNA (서열번호:118); (73) LPHQDTTKND (서열번호:119); (74) LPHQDTTKNT (서열번호:120); (75) LPHQDTTNNA (서열번호:121); (76) LTHQDTTKNA (서열번호:122); (77) LTKDATKTIA (서열번호:123); (78) LTPQDTTKNA (서열번호:124); (79) LVHQDTTKNA (서열번호:125); (80) LAKANQNTPA (서열번호:126); (81) LATTPITKPA (서열번호:127); (82) LATTPIAKPA (서열번호:128); (83) LAIEDHTKSA (서열번호:129); (84) LAQSEHQRPA (서열번호:130); (85) LAKSPNKDNA (서열번호:131); (86) LANQDYTKTA (서열번호:132); (87) LANSTDQTRA (서열번호:133); (88) LALGETTRPA (서열번호:134); (89) LANSTEQTRA (서열번호:135); (90) LAQADTTKNA (서열번호:136); (91) LASKDITKTA (서열번호:137); (92) LASPRHNKKC (서열번호:138); (93) LAHQDTTKTIA (서열번호:139); (94) LAAQGTANL (서열번호:140); (95) VAIEDHTKSA (서열번호:141); 및 (96) LAKANQNTPKNA (서열번호:142).
일부 경우에, 펩타이드 삽입물은 (11) PAPQDTTKKA (서열번호:57)이다. 일부 경우에, 펩타이드 삽입물은 (7) LAPDSTTRSA (서열번호:53)이다.
일부 구현예에서, 당해 rAAV 비리온 캡시드는 상응하는 친계 AAV 캡시드 단백질에 관한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입 외의 임의의 다른 아미노산 치환, 삽입, 또는 결실을 포함하지 않는다. 다른 구현예에서, 당해 rAAV 비리온 캡시드는, 상응하는 친계 AAV 캡시드 단백질에 관한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입 외에, 친계 AAV 캡시드 단백질과 비교하여, 1 내지 약 25개의 아미노산 삽입, 결실, 또는 치환을 포함한다. 예를 들어, 일부 구현예에서, 당해 rAAV 비리온 캡시드는 상응하는 친계 AAV 캡시드 단백질에 관한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입 외에, 친계 AAV 캡시드 단백질과 비교하여 1 내지 약 5, 약 5 내지 약 10, 약 10 내지 약 15, 약 15 내지 약 20, 또는 약 20 내지 약 25개의 아미노산 삽입, 결실, 또는 치환을 포함한다.
일부 경우에, 당해 rAAV 비리온 캡시드는 하기 아미노산 치환: Y273F, Y444F, Y500F, 및 Y730F 중 1, 2, 3, 또는 4개를 포함하지 않는다.
일부 경우에, 당해 변이체 캡시드 폴리펩타이드는, 상기에 기재된 바와 같은 삽입 펩타이드 외에, 하기 아미노산 치환: Y273F, Y444F, Y500F, 및 Y730F 중 1, 2, 3, 또는 4개를 포함한다.
일부 경우에, 당해 rAAV 비리온 캡시드는 키메라성 캡시드이고, 예를 들어, 캡시드는 제1 AAV 혈청형의 AAV 캡시드의 부분 및 제2 혈청형의 AAV 캡시드의 부분을 포함하고; 그리고 상응하는 친계 AAV 캡시드 단백질에 관한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입을 포함한다.
일부 구현예에서, 당해 rAAV 비리온은 하기를 포함한다: 도 4에서 제공된 아미노산 서열에 대해 적어도 약 85%, 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 또는 적어도 약 99%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 캡시드 단백질; 및 상응하는 친계 AAV 캡시드 단백질에 관한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입. 일부 구현예에서, 당해 rAAV 비리온은 하기를 포함한다: 도 4에서 제공된 아미노산 서열에 대해 적어도 약 85%, 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 또는 적어도 약 99%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 캡시드 단백질; 및 도 4에서 묘사된 아미노산 서열에 관한 아미노산 587 내지 588의 사이, 또는 친계 AAV 캡시드 단백질에 관한 상응하는 부위에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입.
일부 구현예에서, 당해 rAAV 비리온은 도 5에서 제시된 아미노산 서열에 대해 적어도 약 85%, 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하고, 굵고 밑줄친 아미노산 사이에 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입을 포함하는 GH 루프를 포함하는 캡시드 단백질을 포함한다.
일부 구현예에서, 당해 rAAV 비리온은 하기를 포함한다: 도 6A-6C에서 제공된 아미노산 서열 중 임의의 하나 에 대해 적어도 약 85%, 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 또는 적어도 약 99%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 캡시드 단백질; 및 AAV2의 아미노산 587 내지 588의 사이, 또는 또 다른 AAV 유전자형에 관한 상응하는 부위에서 약 5개의 아미노산 내지 약 20개의 아미노산 (예를 들어, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 또는 20개의 아미노산; 예를 들어, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 또는 12개의 아미노산)의 삽입. 일부 경우에, 상응하는 삽입 부위는 도 6B에서 볼드체 및 밑줄 친 것으로 나타낸 부위이다.
당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 상기 망막 세포의 감염성과 비교하여, 망막 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 경우에, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 상기 망막 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때, 망막 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 광수용체 세포의 감염성과 비교하여, 광수용체 (간상 또는 원뿔) 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 광수용체 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 광수용체 (간상 또는 원뿔) 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 RGC의 감염성과 비교하여, RGC의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 RGC의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 RGC의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 RPE 세포의 감염성과 비교하여, RPE 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 RPE 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때, RPE 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 뮐러 세포의 감염성과 비교하여, 뮐러 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 뮐러 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 뮐러 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 두극 세포의 감염성과 비교하여, 두극 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 두극 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 두극 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 무축삭 세포의 감염성과 비교하여, 무축삭 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 무축삭 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 무축삭 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 수평 세포의 감염성과 비교하여, 수평 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 수평 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 수평 세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 망막 별아교세포의 감염성과 비교하여, 망막 별아교세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 망막 별아교세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때 망막 별아교세포의 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
일부 경우에, 당해 rAAV 비리온은 ILM을 가로 지르기 위한 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온의 능력과 비교하여, 내경계막 (ILM)을 가로지르기 위해 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 능력을 나타낸다.
일부 경우에, 당해 rAAV 비리온은 유리체내 주사를 통해 투여될 때 ILM을 가로 지르기 위한 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온의 능력과 비교하여, 유리체내 주사를 통해 투여될 때 ILM을 가로지르기 위해 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 능력을 나타낸다.
당해 rAAV 비리온은 ILM를 가로지를 수 있고, 또한 광수용체 세포 및/또는 RPE 세포에 도달하기 위해 뮐러 세포, 무축삭 세포, 등을 포함하는 세포 층을 횡단할 수 있다. 예를 들어, 당해 rAAV 비리온은, 유리체내 주사를 통해 투여될 때, ILM을 가로지를 수 있고, 또한 광수용체 세포 및 또는 RPE 세포에 도딜하기 위해 뮐러 세포, 무축삭 세포, 등을 포함하는 세포 층을 횡단할 수 있다.
일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 ILM을 넘어스는 국재와의 정도와 비교하여, ILM을 넘어서는, 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다. 예를 들어, 일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 RPE 층에 대한 국재화의 정도와 비교하여, 망막 색소 상피 (RPE)에 대한 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다. 또 다른 예로서, 일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 PR 층에 대한 국재화의 정도와 비교하여, 광수용체 (PR) 층에 대한 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다. 또 다른 예로서, 일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 내부 핵 층에 대한 국재화의 정도와 비교하여, 내부 핵 층 에 대한 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다. 또 다른 예로서, 일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 외부 핵 층에 대한 국재화의 정도와 비교하여, 외부 핵 층 에 대한 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다. 또 다른 예로서, 일부 경우에, 당해 rAAV 비리온은, 유리체내로 주사될 때, 상응하는 친계 AAV 캡시드 단백질을 포함하는 유리체내로 주사된 대조군 AAV 비리온에 의한 신경절 세포 층에 대한 국재화의 정도 와 비교하여, 신경절 세포 층 에 대한 적어도 5-배, 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 국재화를 나타낸다.
일부 구현예에서, 당해 rAAV 비리온은 선택적으로 망막 세포를 감염시키고, 예를 들어, 당해 rAAV 비리온은 비-망막 세포, 예를 들어, 눈 외부의 세포 보다 10-배, 15-배, 20-배, 25-배, 50-배, 또는 50-배 초과의 특이성으로 망막 세포를 감염시킨다. 예를 들어, 일부 구현예에서, 당해 rAAV 비리온은 선택적으로 i망막 세포를 감염시키고, 예를 들어, 당해 rAAV 비리온은 비-망막 세포, 예를 들어, 눈 외부의 세포보다 10-배, 15-배, 20-배, 25-배, 50-배, 또는 50-배 초과의 특이성으로 광수용체 세포를 감염시킨다.
일부 구현예에서, 당해 rAAV 비리온은 선택적으로 광수용체 세포를 감염시키고, 예를 들어, 당해 rAAV 비리온은 눈에 존재하는 비-광수용체 세포, 예를 들어, 망막 신경절 세포, 뮐러 세포, 등보다 10-배, 15-배, 20-배, 25-배, 50-배, 또는 50-배 초과의 특이성으로 광수용체 세포를 감염시킨다.
일부 구현예에서, 당해 rAAV 비리온은 to 유리체내 주사를 통해 투여될 때 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 광수용체 세포의 감염성과 비교하여, 유리체내 주사를 통해 투여될 때, 광수용체 세포의 적어도 10-배, 적어도 15-배, 적어도 20-배, 적어도 25-배, 적어도 50-배, 또는 50-배 초과의 증가된 감염성을 나타낸다.
유전자 산물
당해 rAAV 비리온은 유전자 산물 (이종성 유전자 산물)을 인코딩하는 뉴클레오타이드 서열을 포함하는 이종성 핵산을 포함한다. 일부 경우에, 유전자 산물은 폴리펩타이드이다. 일부 경우에, 유전자 산물은 RNA이다. 유전자 산물이 RNA인 경우, 일부 경우에, RNA 유전자 산물은 encode 폴리펩타이드을 인코딩한다. 일부 경우에, 본 개시내용의 rAAV 비리온은 단일 이종성 유전자 산물을 인코딩하는 뉴클레오타이드 서열 을 포함하는 단일 이종성 핵산을 포함한다. 일부 경우에, 본 개시내용의 rAAV 비리온은 2종의 이종성 유전자 산물을 인코딩하는 뉴클레오타이드 서열을 포함하는 단일 이종성 핵산을 포함한다. 일부 경우에, 본 개시내용의 rAAV 비리온은 2종의 이종성 핵산을 포함하되, 이들 각각은 이종성 유전자 산물을 인코딩하는 뉴클레오타이드 서열을 포함한다.
일부 구현예에서, 유전자 산물은 간섭 RNA이다. 일부 구현예에서, 유전자 산물은 압타머이다. 일부 구현예에서, 유전자 산물은 폴리펩타이드이다. 일부 구현예에서, 유전자 산물은 유전자기능의 부위-특이적인 넉다운을 제공하는 부위-특이적인 뉴클레아제이다. 일부 구현예에서, 유전자 산물은 표적 핵산의 변경을 제공하는 RNA-유도된 엔도뉴클레아제이다.
간섭 RNA
유전자 산물이 간섭 RNA (RNAi)인 경우, 적합한 RNAi는 세포에서 세포자멸적 또는 혈관신생 인자의 수준을 감소시키는 RNAi를 포함한다. 예를 들어, RNAi는 또는 촉진하다 세포에서 세포자멸사를 유도하거나 촉진하는 유전자 산물의 수준을 감소시키는 shRNA 또는 siRNA일 수 있다. 유전자 산물이 세포자멸사를 유도하거나 촉진하는 유전자는 본 명세서에서 일명 "세포자멸유도 유전자"이고, 그리고 유전자 (mRNA; 단백질)의 산물은 "세포자멸유도 유전자 산물"로 칭한다. 세포자멸유도 유전자 산물은, 예를 들어, Bax, Bid, Bak, 및 Bad 유전자 산물을 포함한다. 참고, 예를 들어, 미국 특허 번호 7,846,730.
간섭 RNA는 또한 하기에 대항할 수 있다: 혈관신생 생성물, 예를 들어 혈관 내피 성장 인자 (VEGF) (예를 들어, Cand5; 참고, 예를 들어, 미국 특허 공보 번호 2011/0143400; 미국 특허 공보 번호 2008/0188437; 및 Reich 등 (2003) Mol. Vis. 9:210); VEGF 수용체-1 (VEGFR1) (예를 들어, Sirna-027; 참고, 예를 들어, Kaiser 등 (2010) Am. J. Ophthalmol. 150:33; and Shen 등 (2006) Gene Ther. 13:225); 또는 VEGF 수용체-2 (VEGFR2) (Kou 등 (2005) Biochem. 44:15064). 또한 하기를 참조한다: U.S. 특허 번호 6,649,596, 6,399,586, 5,661,135, 5,639,872, 내지 5,639,736; 및 U.S. 특허 번호 7,947,659 및 7,919,473.
압타머
유전자 산물이 압타머인 경우, 관심 예시적인 압타머는 VEGF에 대항하는 압타머를 포함한다. 참고, 예를 들어, Ng 등 (2006) Nat. Rev. Drug Discovery 5:123; 및 Lee 등 (2005) Proc. Natl. Acad. Sci. USA 102:18902. 예를 들어, VEGF 압타머는 the 뉴클레오타이드 서열 5'-cgcaaucagugaaugcuuauacauccg-3' (서열번호://)를 포함할 수 있다. 또한 혈소판-유래된 성장 인자 (PDGF)-특정 압타머, 예를 들어, E10030가 사용하기에 적합하다; 참고, 예를 들어, Ni 및 Hui (2009) Ophthalmologica 223:401; 및 Akiyama 등 (2006) J. Cell Physiol. 207:407).
폴리펩타이드
유전자 산물이 폴리펩타이드인 경우, 폴리펩타이드는 일반적으로 망막 세포의 기능을 향상시키는 폴리펩타이드, 예를 들어, 간상체 또는 원추 광수용체 세포, 망막 신경절 세포, 뮐러 세포, 두극 세포, 무축삭 세포, 수평 세포, 또는 망막 색소 상피 세포의 기능이다. 예시적인 폴리펩타이드는 하기를 포함한다: 신경보호성 폴리펩타이드 (예를 들어, 신경교 세포 유래된 신경친화성 인자 (GDNF), 섬모 신경친화성 인자 (CNTF), 뉴로트로핀-4 (NT4), 신경 성장 인자 (NGF), 및 뉴르투린 (NTN)); 항-혈관신생 폴리펩타이드 (예를 들어, 가용성 VEGF 수용체; VEGF-결합 항체; VEGF-결합 항체 단편 (예를 들어, 단일 사슬 항-VEGF 항체); 엔도스타틴; 텀스타틴; 안지오스타틴; 가용성 Flt 폴리펩타이드 (Lai 등 (2005) Mol. Ther. 12:659); 가용성 Flt 폴리펩타이드를 포함하는 Fc 융합 단백질 (참고, 예를 들어, Pechan 등 (2009) Gene Ther. 16:10); 안료 상피-유래 인자 (PEDF); 가용성 Tie-2 수용체; 등); 메탈로프로테이나제-3 (TIMP-3)의 조직 억제제; 광-반응성 옵신, 예를 들어, 로돕신; 항-세포자멸적 폴리펩타이드 (예를 들어, Bcl-2, Bcl-Xl; XIAP); 및 기타 동종의 것. 적합한 폴리펩타이드는, 비제한적으로, 하기를 포함한다: 신경교 유래된 신경친화성 인자 (GDNF); 섬유모세포 성장 인자; 섬유모세포 성장 인자 2; 뉴르투린 (NTN); 섬모 신경친화성 인자 (CNTF); 신경 성장 인자 (NGF); 뉴로트로핀-4 (NT4); 뇌 유래된 신경친화성 인자 (BDNF; 예를 들어, 도 7B에서 묘사된 아미노산 서열 (서열번호:11)의 약 200개의 아미노산 내지 247개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드); 표피 성장 인자; 로돕신; 세포자멸사의 X-연결된 억제제; 및 소닉 헤지혹.
적합한 광-반응성 옵신은, 예를 들어, 하기에 기재된 바와 같은 광-반응성 옵신을 포함한다: 미국 특허 공보 번호 2007/0261127 (예를 들어, 채널로돕신-2; ChR2; Chop2); 미국 특허 공보 번호 2001/0086421; 미국 특허 공보 번호 2010/0015095; 미국 특허 공보 번호 2016/0002302; 미국 특허 공보 번호 2013/0347137; 미국 특허 공보 번호 2013/0019325; 및 Diester 등 (2011) Nat. Neurosci. 14:387. 참고, Thyagarajan 등 (2010) J Neurosci. 30(26):8745-8758; Lagali 등 (2008) Nat Neurosci. 11(6):667-675; Doroudchi 등 (2011) Mol Ther. 19(7):1220-1229; Henriksen 등 (2014) J. Ophthalmic Vis. Res. 9:374; Tomita 등 (2014) Mol. Ther. 22:1434.
적합한 폴리펩타이드는 광-관문 이온 채널 폴리펩타이드를 포함한다. 참고, 예를 들어, Gaub 등 (2014) Proc. Natl. Acad. Sci. USA 111:E5574. 예를 들어, 적합한 폴리펩타이드는 광-관문 이온요구성 글루타메이트 수용체 (LiGluR)이다. 망막 신경절 세포 및 ON-두극 세포 중 LiGluR의 발현은, 광이성질체성 화합물의 존재에서, 광에 대한 세포 반응성을 부여한다. LiGluR은 L439C 치환을 포함한다; 참고, Caporale 등 (2011) Mol Ther. 19:1212-1219; Volgraf 등 (2006) Nat Chem Biol. 2:47-52; 및 Gorostiza 등 (2007) Proc Natl Acad Sci USA. 104:10865-10870. 광이성질체성 화합물은, 예를 들어, 460 nm에서의 피크 효율 (MAG0460)을 갖는 말레이미드-아조벤젠-글루타메이트 0을 포함한다. MAG0460는 하기 구조를 갖는다:
적합한 폴리펩타이드는 또한 레티노쉬신 (예를 들어, 도 7A에서 묘사된 아미노산 서열 (서열번호:10)의 약 200개의 아미노산 내지 224개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드를 포함한다. 적합한 폴리펩타이드는, 예를 들어, 하기를 포함한다: 색소성 망막염 GTPase 조절자 (RPGR)-상호작용 단백질-1 (참고, 예를 들어, 유전자은행 수탁 번호 Q96KN7, Q9EPQ2, 및 Q9GLM3) (예를 들어, 도 7F에서 묘사된 아미노산 서열 (서열번호:15)의 약 1150개의 아미노산 내지 약 1200개의 아미노산, 또는 약 1200개의 아미노산 내지 1286개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드; 페리페린-2 (Prph2) (참고, 예를 들어, 유전자은행 수탁 번호 NP_000313 (예를 들어, 도 7D에서 묘사된 아미노산 서열 (서열번호:13)의 약 300개의 아미노산 내지 346개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드; 및 Travis 등 (1991) Genomics 10:733); 페리페린 (예를 들어, 도 7E에서 묘사된 아미노산 서열 (서열번호:14)의 약 400개의 아미노산 내지 약 470개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드; 망막 색소 상피-특이 단백질 (RPE65), (예를 들어, 도 7C에서 묘사된 아미노산 서열 (서열번호:12)의 약 200개의 아미노산 내지 247개의 아미노산의 인접 스트레치에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드) (참고, 예를 들어, 유전자은행 AAC39660; 및 Morimura 등 (1998) Proc. Natl. Acad. Sci. USA 95:3088); 간상체-유래된 추상체 생존력 인자 (RdCVF) (예를 들어, 도 7H, 7I, 및 7J 중 임의의 하나에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드; 맥락막결손 (예를 들어, 도 7G에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드); 색소성 망막염 GTPase 조절자 (RPGR) (예를 들어, 도 7S-7V 중 하나에 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함하는 폴리펩타이드); 및 기타 동종의 것. 예를 들어, 일부 경우에, 적합한 RPGR 폴리펩타이드는 도 7S에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다. 또 다른 예로서, 일부 경우에, 적합한 RPGR 폴리펩타이드는 도 7T에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다. 예로서, 일부 경우에, 적합한 RPGR 폴리펩타이드는 도 7U에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다. 예로서, 일부 경우에, 적합한 RPGR 폴리펩타이드는 도 7V에서 묘사된 아미노산 서열에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다.
적합한 폴리펩타이드는 또한 하기를 포함한다: CHM (맥락막결손 (Rab 호송 단백질 1 (REP1))), 결함있거나 없을 때, 맥락막결손을 야기하는 폴리펩타이드 (참고, 예를 들어, Donnelly 등 (1994) Hum. Mol. Genet. 3:1017; 및 van Bokhoven 등 (1994) Hum. Mol. Genet. 3:1041); 및 크럼버스 동족체 1 (CRB1), 결함있거나 없을 때, 레버 선천성 흑내장 및 색소성 망막염을 야기하는 폴리펩타이드 (참고, 예를 들어, den Hollander 등 (1999) Nat. Genet. 23:217; 및 유전자은행 수탁 번호 CAM23328). 예를 들어, 적합한 REP1 폴리펩타이드는 도 7G에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다.
적합한 폴리펩타이드는 간상체 cGMP-특정 3',5'-환형 포스포디에스테라제 하위단위 알파 (PDE6α), 간상체 cGMP-특정 3',5'-환형 포스포디에스테라제 하위단위 베타 동형체 1 (PDE6β 동형체 1), 간상체 cGMP-특정 3',5'-환형 포스포디에스테라제 하위단위 베타 동형체 2 (PDE6β 동형체 2), 간상체 cGMP-특정 3',5'-환형 포스포디에스테라제 하위단위 베타 동형체 3 (PDE6β 동형체 3)를 포함한다. 예를 들어, 적합한 PDE6α 폴리펩타이드는 도 7K에서 묘사된 아미노산 서열 세트에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다. 또 다른 예로서, 적합한 PDE6β6 동형체 1 폴리펩타이드는 도 7L에서 묘사된 아미노산 서열 세트에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다. 또 다른 예로서, 적합한 PDE6β6 동형체 2 폴리펩타이드는 도 7M에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다. 또 다른 예로서, 적합한 PDE6β6 동형체 3 폴리펩타이드는 도 7N에서 묘사된 아미노산 서열 세트에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다.
적합한 폴리펩타이드는 또한, 결함있거나 없을 때, 색맹으로 이어지는 폴리펩타이드 를 포함하고, 그와 같은 폴리펩타이드는, 예를 들어, 하기를 포함한다: 원추 광수용체 cGMP-관문 채널 하위단위 알파 (CNGA3) (참고, 예를 들어, 유전자은행 수탁 번호 NP_001289; 및 Booij 등 (2011) Ophthalmology 118:160-167); 원추 광수용체 cGMP-게이팅된 양이온 통로 베타-하위단위 (CNGB3) (참고, 예를 들어, Kohl 등(2005) Eur J Hum Genet. 13(3):302); 구아닌 뉴클레오타이드 결합 단백질 (G 단백질), 알파 형질도입 활성 폴리펩타이드 2 (GNAT2) (ACHM4); 및 ACHM5; 및 결함있거나 없을 때, 다양한 형태의 색상 시각상실로 이어지는 폴리펩타이드 (예를 들어, L-옵신, M-옵신, 및 S-옵신). 참고 Mancuso 등 (2009) Nature 461(7265):784-787.
예를 들어, 적합한 CNGA3 (ACHM2 로도 공지됨) 동형체 1 폴리펩타이드는 도 7O에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다. 또 다른 예로서, 적합한 CNGA3 (ACHM2로도 공지됨) 동형체 2 폴리펩타이드는 도 7P에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다.
또 다른 예로서, 적합한 CNGB3 (로도 공지된다 ACHM3) 폴리펩타이드는 도 7Q에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다. 또 다른 예로서, GNAT2 (로도 공지된다 ACHM4)는 도 7R에서 묘사된 아미노산 서열 세트 에 대해 적어도 약 90%, 적어도 약 95%, 적어도 약 98%, 적어도 약 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산을 포함할 수 있다.
부위-특이적인 엔도뉴클레아제
일부 경우에, 관심 유전자 산물은 유전자 기능의 부위-특이적인 넉다운을 제공하는 부위-특이적인 엔도뉴클레아제이되, 예를 들어, 상기 엔도뉴클레아제는 망막 질환과 관련된 대립유전자를 녹아웃시킨다. 예를 들어, 우세한 대립유전자는, 야생형일 때, 망막 구조 단백질이고/거나 정상 망막 기능을 제공하는 유전자의 결함있는 복제물 을 인코딩하는 경우, 부위-특이적인 엔도뉴클레아제는 결함있는 대립유전자를 표적으로 할 수 있고 결함있는 대립유전자를 녹아웃시킬 수 있다. 일부 경우에, 부위-특이적인 엔도뉴클레아제는 RNA-유도된 엔도뉴클레아제이다.
결함있는 대립유전자를 녹아웃시키는 것 외에, 부위-특이적인 뉴클레아제는 또한 결함있는 대립유전자에 의해 인코딩되는 단백질의 기능적 복제물을 인코딩하는 공여체 DNA와의 상동성 재조합을 자극하기 위해 사용될 수 있다. 따라서, 예를 들어, 당해 rAAV 비리온은 c 결함있는 대립유전자를 녹아웃시키는 부위-특이적인 엔도뉴클레아제 둘 모두를 전달하기 위해 사용될 수 있고, 결함있는 대립유전자의 기능적 복제물을 전달하여, 결함있는 대립유전자의 회복을 초래하고, 그렇게 함으로써 기능적 망막 단백질 (예를 들어, 기능적 레티노쉬신, 기능적 RPE65, 기능적 페리페린, 등)의 생산을 제공하기 위해 사용될 수 있다. 참고, 예를 들어, Li 등 (2011) Nature 475:217. 일부 구현예에서, 당해 rAAV 비리온은 부위-특이적인 엔도뉴클레아제를 인코딩하는 이종성 뉴클레오타이드 서열; 및 결함있는 대립유전자의 기능적 복제물을 인코딩하는 이종성 뉴클레오타이드 서열을 포함하되, 상기 기능적 복제물은 기능적 망막 단백질을 인코딩한다. 기능적 망막 단백질은, 예를 들어, 레티노쉬신, RPE65, 색소성 망막염 GTPase 조절자 (RGPR)-상호작용 단백질-1, 페리페린, 페리페린-2, RdCVF, 및 기타 동종의 것을 포함한다.
사용하는데 적합한 부위-특이적인 엔도뉴클레아제는, 예를 들어, 아연 핑거 뉴클레아제 (ZFNs); 메가뉴클레아제; 및 전사 활성제-유사 효과기 뉴클레아제 (TALENs)을 포함하되, 그와 같은 부위-특이적인 엔도뉴클레아제는 비-자연 발생적이고, 변형되어 특이 유전자를 표적으로 한다. 그와 같은 부위-특이적인 뉴클레아제는 조작되어 게놈 내의 특정 위치를 절단할 수 있고, 비-상동성 말단 연결는 그 다음 몇 개의 뉴클레오타이드를 삽입 또는 결실시키면서 중단을 복구할 수 있다. 그와 같은 부위-특이적인 엔도뉴클레아제 (또한 일명 "INDELs")은 그 다음 단백질을 프렘임 밖으로 던지고 효과적으로 유전자를 녹아웃시킨다. 참고, 예를 들어, 미국 특허 공보 번호 2011/0301073. 적합한 부위-특이적인 엔도뉴클레아제는 조작된 메가뉴클레아제 재-조작된 귀소 엔도뉴클레아제를 포함한다. 적합한 엔도뉴클레아제는 I-Tevl 뉴클레아제를 포함한다. 적합한 메가뉴클레아제는 I-Sce1을 포함한다 (참고, 예를 들어, Bellaiche 등 (1999) Genetics 152:1037); 및 I-Cre1 (참고, 예를 들어, Heath 등 (1997) Nature Sructural Biology 4:468).
RNA-유도된 엔도뉴클레아제
일부 경우에, 유전자 산물은 RNA-유도된 엔도뉴클레아제이다. 일부 경우에, 유전자 산물은 RNA-유도된 엔도뉴클레아제를 인코딩하는 뉴클레오타이드 서열을 포함하는 RNA이다. 일부 경우에, 유전자 산물은 가이드 RNA, 예를 들어, 단일-가이드 RNA이다. 일부 경우에, 유전자 산물은 하기이다: 1) 가이드 RNA; 및 2) RNA-유도된 엔도뉴클레아제. 가이드 RNA는 하기를 포함할 수 있다: a) RNA-유도된 엔도뉴클레아제에 결합하는 단백질-결합 영역; 및 b) 표적 핵산에 결합하는 영역. RNA-유도된 엔도뉴클레아제는 또한 본 명세서에서 일명 "게놈 편집 뉴클레아제"이다.
적합한 게놈 편집 뉴클레아제의 예는 CRISPR/Cas 엔도뉴클레아제 (예를 들어, 부류 2 CRISPR/Cas 엔도뉴클레아제 예컨대 유형 II, 유형 V, 또는 유형 VI CRISPR/Cas 엔도뉴클레아제)이다. 따라서, 게놈 표적화 조성물은 CRISPR/Cas 엔도뉴클레아제 (예를 들어, 부류 2 CRISPR/Cas 엔도뉴클레아제 예컨대 유형 II, 유형 V, 또는 유형 VI CRISPR/Cas 엔도뉴클레아제)를 포함할 수 있다. 일부 경우에, 게놈 표적화 조성물은 부류 2 CRISPR/Cas 엔도뉴클레아제를 포함한다. 일부 경우에, 게놈 표적화 조성물은 부류 2 유형 II CRISPR/Cas 엔도뉴클레아제 (예를 들어, Cas9 단백질) 를 포함한다. 일부 경우에, 게놈 표적화 조성물은 부류 2 유형 V CRISPR/Cas 엔도뉴클레아제 (예를 들어, Cpf1 단백질, C2c1 단백질, 또는 C2c3 단백질) 를 포함한다. 일부 경우에, 게놈 표적화 조성물은 부류 2 유형 VI CRISPR/Cas 엔도뉴클레아제 (예를 들어, C2c2 단백질) 를 포함한다.
일부 경우에, 게놈 편집 뉴클레아제는 이종성 폴리펩타이드 (또한 일명 "융합 파트너")에 융합된 융합 단백질이다. 일부 경우에, 게놈 편집 뉴클레아제는 세포하 국재화를 제공하는 아미노산 서열 (융합 파트너)에 융합되고, 즉, 융합 파트너는 세포하 국재화 서열 (예를 들어, 핵, 2종 이상의 NLSs, 3 또는 그 초과 NLSs, 등을 표적으로 하기 위한 하나 이상의 핵 국재화 신호 (NLSs))이다.
일부 경우에, 게놈-편집 엔도뉴클레아제는 유형 II CRISPR/Case 엔도뉴클레아제이다. 일부 경우에, 게놈-편집 엔도뉴클레아제는 Cas9 폴리펩타이드이다. Cas9 단백질은 Cas9 가이드 RNA의 단백질-결합 세그먼트와의 회합 덕분에 표적 핵산 서열 (예를 들어, 염색체 서열 또는 염색체외 서열, 예를 들어, 에피솜 서열, 미니서클 서열, 미토콘드리아 서열, 엽록체 서열, 등) 내의 표적 부위 (예를 들어, 표적 부위에서 안정화됨)로 유도된다. 일부 경우에, Cas9 폴리펩타이드는 도 8A에서 묘사된 연쇄상구균 파이오제네스 Cas9에 대해 적어도 50%, 적어도 60%, 적어도 70%, 적어도 80%, 적어도 90%, 적어도 95%, 적어도 98%, 적어도 99%, 또는 99% 초과의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다. 일부 경우에, 본 개시내용의 조성물 또는 방법에서 사용된 Cas9 폴리펩타이드는 스타필로코쿠스 아우레스 Cas9 (saCas9) 폴리펩타이드이다. 일부 경우에, saCas9 폴리펩타이드는 도 8B에서 묘사된 saCas9 아미노산 서열에 대해 적어도 85%, 적어도 90%, 적어도 95%, 적어도 98%, 적어도 99%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다.
일부 경우에, 적합한 Cas9 폴리펩타이드는 고-충실도 (HF) Cas9 폴리펩타이드이다. Kleinstiver 등 (2016) Nature 529:490. 예를 들어, 도 8A에서 묘사된 아미노산 서열의 아미노산 N497, R661, Q695, 및 Q926은 예를 들어, 알라닌으로 치환된다. 예를 들어, HF Cas9 폴리펩타이드는 도 8A에서 묘하된 아미노산 서열 에 대해 적어도 90%, 적어도 95%, 적어도 98%, 적어도 99%, 또는 100%의 아미노산 서열을 갖는 아미노산 서열을 포함할 수 있되, 상기 아미노산 N497, R661, Q695, 및 Q926은 예를 들어, 알라닌으로 치환된다.
일부 경우에, 적합한 Cas9 폴리펩타이드는 변경된 PAM 특이성을 나타낸다. 참고, 예를 들어, Kleinstiver 등 (2015) Nature 523:481.
일부 경우에, 게놈-편집 엔도뉴클레아제는 유형 V CRISPR/Cas 엔도뉴클레아제이다. 일부 경우에 유형 V CRISPR/Cas 엔도뉴클레아제는 Cpf1 단백질이다. 일부 경우에, Cpf1 단백질은 도 8C에서 묘사된 Cpf1개의 아미노산 서열에 대해 적어도 30%, 적어도 35%, 적어도 40%, 적어도 45%, 적어도 50%, 적어도 55%, 적어도 60%, 적어도 65%, 적어도 70%, 적어도 75%, 적어도 80%, 적어도 85%, 적어도 90%, 적어도 95%, 적어도 90%, 또는 100%의 아미노산 서열 동일성을 갖는 아미노산 서열을 포함한다.
부류 2 CRISPR/Cas 엔도뉴클레아제 (예를 들어, Cas9 단백질; 유형 V 또는 유형 VI CRISPR/Cas 단백질; Cpf1 단백질; 등)에 결합하고 복합체를 표적 핵산 내의 특정 위치로 표적화하는 핵산은 본 명세서에서 일명 "가이드 RNA" 또는 "CRISPR/Cas 가이드 핵산" 또는 "CRISPR/Cas 가이드 RNA"이다. 가이드 RNA는 표적 핵산의 서열에 대해 상보적인 뉴클레오타이드 서열인 가이드 서열 (또한 본 명세서에서 일명 표적화 서열)을 포함하는 표적화 분절을 포함함으로써 복합체 (RNP 복합체)에 대해 표적 특이성을 제공한다.
일부 경우에, 가이드 RNA는 2개의 별개의 핵산 분자: "활성제" 및 "타겟터"를 포함하고, 본 명세서에서 일명 "이중 가이드 RNA", "이중-분자 가이드 RNA", "2-분자 가이드 RNA", 또는 "dgRNA"이다. 일부 경우에, 가이드 RNA는 하나의 분자 (예를 들어, 일부 부류 2 CRISPR/Cas 단백질에 대해, 상응하는 가이드 RNA는 단일 분자이고; 그리고 일부 경우에, 활성제 및 타겟터는, 예를 들어, 개입 뉴클레오타이드를 통해 서로 공유결합됨)이고, 그리고 가이드 RNA는 "단일 가이드 RNA", "단일-분자 가이드 RNA", "1-분자 가이드 RNA" 또는 간단히 "sgRNA"로 칭한다.
유전자 산물이 RNA-유도된 엔도뉴클레아제이거나 RNA-유도된 엔도뉴클레아제 및 가이드 RNA 둘 모두인 경우, 유전자 산물은 표적 핵산을 변형시킬 수 있다. 일부 경우에, 예를 들어, 표적 핵산이 결함있는 대립유전자에서 유해한 돌연변이 (예를 들어, 망막 세포 표적 핵산에서 유해한 돌연변이)를 포함하는 경우, RNA-유도된 엔도뉴클레아제/가이드 RNA 복합체는, 유해한 돌연변이 (예를 들어, 결함있는 대립유전자에 의해 인코딩되는 단백질의 기능적 복제물을 인코딩하는 뉴클레오타이드 서열를 공여체 핵산)을 수정하는 뉴클레오타이드 서열을 포함하는 공여체 핵산과 함께, 예를 들어, 상동 지정 복구 (HDR)를 통해 유해한 돌연변이를 수정하기 위해 사용될 수 있다.
일부 경우에, 유전자 산물은 RNA-유도된 엔도뉴클레아제 및 2 별개의 sgRNA이되, 상기 2개의 별개 sgRNA는 비-상동성 말단 연결 (NHEJ)를 통해 표적 핵산의 결실을 제공한다.
본 개시내용은 개체의 망막 세포에서 표적 핵산을 변형시키는 방법을 제공하되, 상기 표적 핵산은 유해한 돌연변이를 포함하고, 상기 방법은 상기 개체에게 (예를 들어, 안구내에 의해; 유리체내; 등 투여) 본 개시내용의 rAAV 비리온을 투여하는 것을 포함하고, 상기 rAAV 비리온은 하기를 포함하는 이종성 핵산을 포함한다: i) RNA-유도된 엔도뉴클레아제를 인코딩하는 뉴클레오타이드 서열 (예를 들어, Cas9 엔도뉴클레아제); ii) 상기 표적 핵산에 대해 상보적인 뉴클레오타이드 서열을 포함하는 sgRNA 을 인코딩하는 뉴클레오타이드 서열; 및 iii) 유해한 돌연변이를 정정하는 뉴클레오타이드 서열을 포함하는 공여체 DNA 템플레이트를 인코딩하는 뉴클레오타이드 서열. rAAV 비리온의 투여는 HDR에 의해 상기 표적 핵산에서의 유해한 돌연변이의 정정을 초래한다.
본 개시내용은 개체의 망막 세포에서 표적 핵산을 변형시키는 방법을 제공하되, 상기 표적 핵산은 유해한 돌연변이를 포함하고, 상기 방법은 상기 개체에게 (예를 들어, 안구내에 의해; 유리체내; 등 투여) 본 개시내용의 rAAV 비리온을 투여하는 것을 포함하고, 상기 rAAV 비리온은 하기를 포함하는 이종성 핵산을 포함한다: i) RNA-유도된 엔도뉴클레아제를 인코딩하는 뉴클레오타이드 서열 (예를 들어, Cas9 엔도뉴클레아제); ii) 상기 표적 핵산에서 제1 서열에 대해 상보적인 뉴클레오타이드 서열을 포함하는 제1 sgRNA 을 인코딩하는 뉴클레오타이드 서열; 및 iii) 상기 표적 핵산에서 제2 서열에 대해 상보적인 뉴클레오타이드 서열을 포함하는 제2 sgRNA 을 인코딩하는 뉴클레오타이드 서열. rAAV 비리온의 투여는 NHEJ에 의해 상기 표적 핵산에서의 유해한 돌연변이의 절개를 초래한다.
조절 서열
일부 경우에, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 전사 조절 요소 에 작동가능하게 연결된다. 예를 들어, 일부 경우에, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 항시성 프로모터 에 작동가능하게 연결된다. 다른 사례에서, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 유도성 프로모터 에 작동가능하게 연결된다. 일부 사례에서, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 조직-특정 또는 세포 유형-특정 조절 인자 에 작동가능하게 연결된다. 예를 들어, 일부 사례에서, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 망막 세포 특이적 프로모터 에 작동가능하게 연결된다. 예를 들어, 일부 사례에서, 관심 유전자 산물을 인코딩하는 뉴클레오타이드 서열은 광수용체-특정 조절 인자 (예를 들어, 광수용체-특정 프로모터), 예를 들어, 광수용체 세포에서 작동가능하게 연결된 유전자의 선택적 발현을 부여하는 조절 인자 에 작동가능하게 연결된다. 적합한 광수용체-특정 조절 인자는, 예를 들어, 하기를 포함한다: 로돕신 프로모터; 로돕신 키나제 프로모터 (Young 등 (2003) Ophthalmol. Vis. Sci. 44:4076); 베타 포스포디에스테라제 유전자 프로모터 (Nicoud 등 (2007) J. Gene Med. 9:1015); 색소성 망막염 유전자 프로모터 (Nicoud 등 (2007) supra); 인터포토리셉터 레티노이드-결합 단백질 (IRBP) 유전자 증진제 (Nicoud 등 (2007) supra); IRBP 유전자 프로모터 (Yokoyama 등 (1992) Exp Eye Res. 55:225).
약제학적 조성물
본 개시내용은 하기를 포함하는 약제학적 조성물을 제공한다: a) 당해 rAAV 비리온, 상기에 기재된 바와 같이; 및 b) 약제학적으로 허용가능한 담체, 희석제, 부형제, 또는 완충액. 일부 구현예에서, 약제학적으로 허용가능한 담체, 희석제, 부형제, 또는 완충액은 인간에 사용하기에 적합하다.
그와 같은 부형제, 캐리어, 희석제, 및 완충액은 과도한 독성 없이투여될 수 있는 임의의 약제학적 제제를 포함한다. 약제학적으로 허용가능한 부형제는, 비제한적으로, 하기를 포함한다: 액체 예컨대 물, 염수, 글리세롤 및 에탄올. 약제학적으로 허용가능한 염는 본 명세서에 내에 포함될 수 있고, 그 예는 무기산 염 예컨대 하이드로클로라이드, 하이드로브로마이드, 포스페이트, 설페이트, 및 기타 동종의 것; 및 유기 산의 염 예컨대 아세테이트, 프로피오네이트, 말로네이트, 벤조에이트, 및 기타 동종의 것이다. 추가로, 보조 물질, 예컨대 습윤 또는 유화제, pH 완충 물질, 및 등은, 그와 같은 비히클 내에 존재할 수 있다. 다양한 약제학적으로 허용가능한 부형제는 당해 기술에 공지되어 있고 본 명세서에서 상세히 논의될 필요는 없다. 약제학적으로 허용가능한 부형제는 예를 들어 하기를 포함하는 다양한 공보에 충분히 기재되었다: A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel 등, eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe 등, eds., 제3판. Amer. Pharmaceutical Assoc.
유전자 산물을 망막 세포에 전달하는 방법 및 치료 방법
본 개시내용은 개체에서 유전자 산물을 망막 세포에 전달하는 방법을 제공하고, 상기 방법은 상기 개체에게 상기에 기재된 바와 같은 당해 rAAV 비리온을 투여하는 것을 포함한다. 유전자 산물은 상기에 기재된 바와 같이, 폴리펩타이드 또는 간섭 RNA (예를 들어, shRNA, siRNA, 및 기타 동종의 것), 압타머, 또는 부위-특이적인 엔도뉴클레아제 (예를 들어, RNA-유도된 엔도뉴클레아제)일 수 있다. 유전자 산물을 망막 세포에 전달하는 것은 망막 질환의 치료를 제공할 수 있다. 망막 세포는 광수용체, 망막 신경절 세포, 뮐러 세포, 두극 세포, 무축삭 세포, 수평 세포, 또는 망막 색소 상피 세포일 수 있다. 일부 경우에, 망막 세포는 광수용체 세포, 예를 들어, 간상 또는 원뿔 세포이다.
본 개시내용은 망막 세포에서 표적 핵산을 변형시키는 방법을 제공하고, 상기 방법은 망막 세포를 하기와 접촉시키는 것을 포함한다: 1) 본 개시내용의 rAAV 비리온으로서, 가이드 RNA에 결합하는 RNA-유도된 엔도뉴클레아제를 인코딩하는 뉴클레오타이드 서열을 포함하는 이종성 핵산을 포함하는 상기 rAAV 비리온; 및 2) 가이드 RNA. 본 개시내용은 망막 세포에서 표적 핵산을 변형시키는 방법을 제공하고, 상기 방법은 망막 세포를 본 개시내용의 rAAV 비리온과 접촉시키는 것을 포함하되, 상기 rAAV 비리온은 하기를 인코딩하는 뉴클레오타이드 서열을 포함하는 이종성 핵산을 포함한다: i) 가이드 RNA에 결합하는 RNA-유도된 엔도뉴클레아제; 및 ii) 가이드 RNA. 일부 경우에, 상기 방법은 망막 세포를 공여체 DNA 템플레이트와 접촉시키는 것을 포함한다. 일부 경우에, RNA-유도된 엔도뉴클레아제는 Cas9 폴리펩타이드이다. 일부 경우에, 가이드 RNA는 단일-가이드 RNA이다.
본 개시내용은 안구 질환 (예를 들어, 망막 질환)을 치료하는 방법을 제공하고, 상기 방법은 그것이 필요한 개체에게 상기에 기재된 바와 같은 당해 rAAV 비리온의 유효량을 투여하는 것을 포함한다. 당해 rAAV 비리온은 안구내 주사를 통해, 유리체내 주사에 의해, 또는 임의의 다른 편리한 방식 또는 투여 경로에 의해 투여될 수 있다. 다른 편리한 방식 또는 투여 경로는, 예를 들어, 정맥내, 비강내, 등을 포함한다.
"치료적 유효량"은 실험과정 및/또는 임상시험을 통해 결정될 수 있는 상대적으로 넓은 범위 내에 있을 것이다. 예를 들어, 생체내 주사, 즉, 눈에 직접 주사에 대해, 치료적으로 효과적인 용량은 대략 약 106 내지 약 1015의 rAAV 비리온, 예를 들어, 약 108 내지 1012 rAAV 비리온일 것이다. 시험관내 형질도입에 대해, 세포에 전달될 rAAV 비리온의 유효량은 대략 약 108 내지 약 1013의 rAAV 비리온일 것이다. 다른 효과적인 투약량은 용량 반응 곡선을 확립하는 일상적인 시험을 통해 당해 분야의 숙련가에 의해 쉽게 확립될 수 있다.
일부 구현예에서, 1회 초과의 투여 (예를 들어, 2, 3, 4 또는 그 초과 투여)는 유전자 발현의 원하는 수준을 달성하기 위해 이용될 수 있다. 일부 경우에, 1회 초과의 투여는 다양한 간격, 예를 들어, 매일, 매주, 매달 2회, 매월, 매 3 개월, 매 6 개월, 매년, 등으로 투여된다. 일부 경우에, 다중 투여는 1 개월 내지 2 개월, 2 개월 내지 4 개월, 4 개월 내지 8 개월, 8 개월 내지 12 개월, 1 년 내지 2 년, 2 년 내지 5 년, 또는 5 년 초과의 일정 기간에 걸쳐 투여된다.
본 발명을 사용하여 치료될 수 있는 안구 질환은, 비제한적으로, 하기를 포함한다: 급성 황반 신경망막증; 베체트병; 맥락막 신생혈관형성; 당뇨병성 포도막염; 히스토플라마증; 황반 변성, 예컨대 급성 황반 변성, 비-삼출성 연령 관련 황반 변성 및 삼출성 연령 관련 황반 변성; 부종, 예컨대 황반 부종, 포낭 황반 부종 및 당뇨병성 황반 부종; 다초점 맥락막염; 안구 트라우마 which 영향을 주다 안구 뒤쪽 부위 또는 위치; 안구 종양; 망막 장애, 예컨대 중심 망막 정맥 폐색, 당뇨 망막병증 (증식성 당뇨 망막병증 포함), 증식성 유리체망막병증 (PVR), 망막 동맥 폐쇄성 질환, 망막 탈착, 포도막 망막 질환; 교감성 안염; 보고트(Vogt) 고야나기-하라다 (VKH) 증후군; 포도막 확산; 안구 레이저 치료에 의해 야기되거나 영향을 받은 후측 안구 병태; 광역학적 요법에 의해 야기되거나 영향을 받은 후측 안구 병태; 광응고, 방사선 망막증; 망막앞 막 장애; 망막 분지 정맥 폐색; 전측 허혈성 시신경병증; 비-망막증 당뇨병성 망막 기능이상; 망막층간분리; 색소성 망막염; 녹내장; 어셔 증후군, 추상체-간상체 이상증; 스타가르트 질환 (황반 안저); 선천적 황반 변성; 맥락망막 퇴행; 레버 선천성 흑내장; 선천성 정지된 야맹증; 맥락막결손; 바데트-비들 증후군; 황반 모세혈관확장증; 레버 선천성 시신경병증; 미숙아 망막증; 색맹, 적색맹, 녹색맹, 및 청색맹을 포함하는 색각의 장애; 및 비에티 결정 이상증.
핵산 및 숙주세포
본 개시내용은 상기에 기재된 바와 같은 당해 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질을 인코딩하는 뉴클레오타이드 서열을 포함하는 단리된 핵산을 제공하되, 상기 변이체 AAV 캡시드 단백질은 상응하는 친계 AAV 캡시드 단백질에 대한 GH 루프 또는 루프 IV에서 약 5개의 아미노산 내지 약 20개의 아미노산의 삽입을 포함하고, 그리고 상기 변이체 캡시드 단백질은, AAV 비리온 내에서 존재할 때, 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의한 상기 망막 세포의 감염성과 비교하여, 망막 세포의 증가된 감염성을 제공한다. 딩해 단리된 핵산는 AAV 벡터, 예를 들어, 재조합 AAV 벡터일 수 있다.
삽입 펩타이드
당해 핵산에 의해 인코딩된 변이체 AAV 캡시드 단백질은 약 5개의 아미노산 내지 약 20개의 아미노산 길이의 삽입 펩타이드를 가지며, 이는 AAV 캡시드의 GH 루프에 삽입된다. 삽입 펩타이드는 5개의 아미노산, 6개의 아미노산, 7개의 아미노산, 8개의 아미노산, 9개의 아미노산, 10개의 아미노산, 11개의 아미노산, 12개의 아미노산, 13개의 아미노산, 14개의 아미노산, 15개의 아미노산, 16개의 아미노산, 17개의 아미노산, 18개의 아미노산, 19개의 아미노산, 또는 20개의 아미노산의 길이를 갖는다. 적합한 삽입 펩타이드는 상기에 기재된 바와 같다. 적합한 삽입 펩타이드는 상기에 기재된 바와 같은 식 I-X 중 임의의 것의 펩타이드를 포함한다.
당해 재조합 AAV 벡터는 상기에 기재된 바와 같은 당해 재조합 AAV 비리온을 생성하기 위해 사용될 수 있다. 따라서, 본 개시내용은, 적합한 세포에 도입될 때, 당해 재조합 AAV 비리온의 생산을 제공할 수 있는 재조합 AAV 벡터를 제공한다.
본 발명은 추가로, 당해 핵산을 포함하는 숙주세포, 예를 들어, 단리된 (유전자 변형된) 숙주세포를 제공한다. 당해 숙주세포는 시험관내 배양에서, 단리된 세포, 예를 들어, 세포일 수 있다. 당해 숙주세포는 아래에 기재된 바와 같은 당해 rAAV 비리온을 생산하는데 유용하다. 당해 숙주세포가 당해 rAAV 비리온을 생산하기 위해 사용되는 경우, "포장 세포"라 칭한다. 일부 구현예에서, 당해 숙주세포는 당해 핵산으로 안정적으로 유전자 변형된다. 다른 구현예에서, 당해 숙주세포는 당해 핵산으로 일시적으로 유전자 변형된다.
당해 핵산은, 전기천공, 인산칼슘 침전, 리포좀-매개된 형질감염, 및 기타 동종의 것을 비제한적으로 포함하는 확립된 기숭을 사용하여 안정적으로 또는 일시적으로 숙주세포에 도입된다. 안정한 전환에 대해, 당해 핵산은 일반적으로 추가로, 선별 마커, 예를 들어, 몇 개의 공지된 선별 마커 중 임의의 것 예컨대 네오마이신 저항, 및 기타 동종의 것을 포함할 것이다.
당해 숙주세포는 당해 핵산을, 다양한 세포, 예를 들어, 쥣과 세포, 및 영장류 세포 (예를 들어, 인간 세포)를 포함하는 포유동물 세포 중 임의의 것에 도입함으로써 생성된다. 적합한 포유동물 세포는, 비제한적으로, 하기를 포함한다: 일차 세포 및 세포주를 포함하되, 적합한 세포주는, 비제한적으로, 하기를 포함한다: 293 세포, COS 세포, HeLa 세포, 베로(Vero) 세포, 3T3 마우스 섬유모세포, C3H10T1/2 섬유모세포, CHO 세포, 및 기타 동종의 것을 포함한다. 적합한 숙주세포의 비-제한적인 예는, 예를 들어, HeLa 세포 (예를 들어, 미국 종균 협회 (ATCC) No. CCL-2), CHO 세포 (예를 들어, ATCC 번호 CRL9618, CCL61, CRL9096), 293 세포 (예를 들어, ATCC No. CRL-1573), 베로(Vero) 세포, NIH 3T3 세포 (예를 들어, ATCC No. CRL-1658), Huh-7 세포, BHK 세포 (예를 들어, ATCC No. CCL10), PC12 세포 (ATCC No. CRL1721), COS 세포, COS-7 세포 (ATCC No. CRL1651), RAT1 세포, 마우스 L 세포 (ATCC No. CCLI.3), 인간 배아 신장 (HEK) 세포 (ATCC No. CRL1573), HLHepG2 세포, 및 기타 동종의 것을 포함한다. 당해 숙주세포는 또한 AAV를 생산하는 곤충 세포 예컨대 Sf9 세포를을 감염시키기 위해 배큘로바이러스를 사용하여 제조될 수 있다 (참고, 예를 들어, 미국 특허 번호 7,271,002; US 특허 출원 12/297,958)
일부 구현예에서, 당해 유전자 변형된 숙주세포는 상기에 기재된 바와 같은 변이체 AAV 캡시드 단백질을 인코딩하는 뉴클레오타이드 서열을 포함하는 핵산 외에, 하나 이상의 AAV rep 단백질을 인코딩하는 뉴클레오타이드 서열을 포함하는 핵산을 포함한다. 다른 구현예에서, 당해 숙주세포는 추가로 rAAV 벡터를 포함한다. rAAV 비리온은 당해 숙주세포를 사용하여 생성될 수 있다. rAAV 비리온을 생성하는 방법은 예를 들어, 미국 특허 공보 번호 2005/0053922 및 미국 특허 공보 번호 2009/0202490에 기재되어 있다.
개시내용의 비제한적인 양태의 예
상기 기재된 본 주제의, 구현예를 포함하는, 양태는, 하나 이상의 다른 양태 또는 구현예로, 단독으로 또는 조합으로 유익할 수 있다. 선행 기재의 제한 없이, 1-34 넘버링된 본 개시내용의 특정 비-제한 양태는 아래 제공된다. 본 개시내용 판독시 종래기술의 숙련가에 명백할 바와 같이, 개별적으로 넘버링된 양태의 각각은 선행 또는 후행 개별적으로 넘버링된 양태의 어느 하나와 조합될 수 있거나 사용될 수 있다. 이것은 양태의 모든 그와 같은 조합을 위하여 뒷받침을 제공하기 위한 의도이고 아래 명확하게 제공된 양태의 조합에 제한되지 않는다:
양태 1. 하기를 포함하는, 재조합 아데노-관련된 바이러스 (rAAV) 비리온: a) 변이체 AAV 캡시드 단백질로서, 상기 변이체 AAV 캡시드 단백질은 상응하는 친계 AAV 캡시드 단백질에 대해 상기 캡시드 단백질 GH 루프에서 약 5개의 아미노산 내지 약 20개의 아미노산의 길이를 갖는 이종성 펩타이드의 삽입을 포함하고, 그리고 상기 변이체 캡시드 단백질은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 대조군 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 증가된 감염성을 부여하는, 변이체 AAV 캡시드 단백질; 및 b) 이종성 유전자 산물을 인코딩하는 뉴클레오타이드 서열을 포함하는 이종성 핵산.
양태 2. 양태 1의 rAAV 비리온으로서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 대조군 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 적어도 5-배수 증가된 감염성을 나타낸다.
양태 3. 양태 1의 rAAV 비리온으로서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 적어도 10-배수 증가된 감염성을 나타낸다.
양태 4. 양태 1 내지 3 중 임의의 하나의 rAAV 비리온으로서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 또는 망막 색소 상피에 대한 국제화의 정도와 비교하여, 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 및 망막 색소 상피 중 하나 이상에 대해 적어도 5-배수 증가된 국재화를 나타낸다.
양태 5. 양태 1 내지 4 중 임의의 하나의 rAAV 비리온으로서, 상기 삽입 부위는 AAV2의 VP1의 아미노산 570 및 611에 상응하는 아미노산, 또는 또 다른 AAV 혈청형의 캡시드 단백질 내의 상응하는 위치 사이에 있다.
양태 6. 양태 1 내지 5 중 임의의 하나의 rAAV 비리온으로서, 상기 삽입 부위는 AAV2의 VP1의 아미노산 587 내지 588에 상응하는 아미노산, 또는 또 다른 AAV 혈청형의 캡시드 단백질 내의 상응하는 위치 사이에 있다.
양태 7. 양태 1 내지 6 중 임의의 하나의 rAAV 비리온으로서, 상기 유전자 산물은 간섭 RNA 또는 압타머이다.
양태 8. 양태 1 내지 6 중 임의의 하나의 rAAV 비리온으로서, 상기 유전자 산물은 폴리펩타이드이다.
양태 9. 양태 8의 rAAV 비리온으로서, 상기 폴리펩타이드는 신경보호성 폴리펩타이드, 항-혈관신생 폴리펩타이드, 또는 망막 세포의 기능을 향상시키는 폴리펩타이드이다.
양태 10. 양태 8의 rAAV 비리온으로서, 상기 폴리펩타이드는 RNA-유도된 엔도뉴클레아제이다.
양태 11. 양태 10의 rAAV 비리온으로서, 상기 RNA-유도된 엔도뉴클레아제는 Cas9 폴리펩타이드이다.
양태 12. 양태 10의 rAAV 비리온으로서, 상기 유전자 산물은 RNA-유도된 엔도뉴클레아제 및 가이드 RNA이다.
양태 13. 양태 1 내지 12 중 임의의 것의 rAAV 비리온으로서, 상기 GH 루프에 삽입된 이종성 펩타이드는 식 I-X 중 임의의 하나의 것이다.
양태 14. 양태 1 내지 12 중 임의의 것의 rAAV 비리온으로서, 상기 이종성 펩타이드는 하기 식 I의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
식 중:
X1는 Leu, Ile, Pro, 또는 Gln이고;
X2는 Ala, Pro, Ser, Asp, Gly, Thr, 또는 Val이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, 또는 Tyr이고;
X4는, 존재한다면, Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, 또는 Arg이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, 또는 Tyr이고;
X6는 Thr, Ala, Gln, Ser, Glu, Pro, 또는 Ile;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, 또는 Cys이고;
X8는 Lys, Ser, Arg, Thr, Ala, Glu, Ile, 또는 Asn이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Phe, Asp, Thr, Val, 또는 Met이다.
양태 15. 양태 14의 rAAV 비리온으로서, 상기 이종성 펩타이드는 하기 아미노산 서열 중 하나를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (23) LAKADETRPA (서열번호:69); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); (32) LAISDQSKPA (서열번호:78); (33) LADATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (35) LAKSDQSRPA (서열번호:81); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (39) LPISDQTKHA (서열번호:85); (40) LPKDATKTIA (서열번호:86); (41) LPPQDTTKNA (서열번호:87); (42) PAPQDTTKNA (서열번호:88); (43) QAHQDTTKNA (서열번호:89); (44) LAHETSPRPA (서열번호:90); (45) LAKSTSTAPA (서열번호:91); (46) LADQDTTKNA (서열번호:92); (47) LAESDQSKPA (서열번호:93); (48) LAHKDTTKNA (서열번호:94); (49) LAHKTQQKM (서열번호:95); (50) LAHQDTTENA (서열번호:96); (51) LAHQDTTINA (서열번호:97); (52) LAHQDTTKKT (서열번호:98); (53) LAHQDTTKND (서열번호:99); (54) LAHQDTTKNT (서열번호:100); (55) LAHQDTTKNV (서열번호:101); (56) LAHQDTTKTM (서열번호:102); (57) LAHQNTTKNA (서열번호:103); (58) LAHRDTTKNA (서열번호:104); (59) LAISDQTNHA (서열번호:105); (60) LAKQKSASTA (서열번호:106); (61) LAKSDQCKPA (서열번호:107); (62) LAKSDQSKPD (서열번호:108); (63) LAKSDQSNPA(서열번호:109); (64) LAKSYQSKPA (서열번호:110); (65) LANQDTTKNA (서열번호:111); (66) LAPQNTTKNA (서열번호:112); (67) LAPSSIQKPA (서열번호:113); (68) LAQQDTTKNA (서열번호:114); (69) LAYQDTTKNA (서열번호:115); (70) LDHQDTTKNA (서열번호:116); (71) LDHQDTTKSA (서열번호:117); (72) LGHQDTTKNA (서열번호:118); (73) LPHQDTTKND (서열번호:119); (74) LPHQDTTKNT (서열번호:120); (75) LPHQDTTNNA (서열번호:121); (76) LTHQDTTKNA (서열번호:122); (77) LTKDATKTIA (서열번호:123); (78) LTPQDTTKNA (서열번호:124); 및 (79) LVHQDTTKNA (서열번호:125).
양태 16. 양태 1 내지 12 중 임의의 것의 rAAV 비리온으로서, 상기 이종성 펩타이드는 하기 식 II의 펩타이드이다:
X1X2X3X4X5X6X7X8X9X10,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala이다.
양태 17. 양태 16의 rAAV 비리온으로서, 상기 펩타이드는 하기 아미노산 서열 중 하나를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); (18) LAVSDSTKAA (서열번호:64); (19) LAAQGTAKKPA (서열번호:65); (20) LAPDQTTRNA (서열번호:66); (21) LAASDSTKAA (서열번호:67); (22) LAPQDTTKNA (서열번호:68); (23) LAKADETRPA (서열번호:69); (24) LAHQDTAKNA (서열번호:70); (25) LAHQDTKKNA (서열번호:71); (26) LAHQDTTKHA (서열번호:72); (27) LAHQDTTKKA (서열번호:73); (28) LAHQDTTRNA (서열번호:74); (29) LAHQDTTNA (서열번호:75); (30) LAHQGTTKNA (서열번호:76); (31) LAHQVTTKNA (서열번호:77); (32) LAISDQSKPA (서열번호:78); (33) LADATKTA (서열번호:79); (34) LAKDTTKNA (서열번호:80); (35) LAKSDQSRPA (서열번호:81); (36) LAPQDTKKNA (서열번호:82); (37) LATSDSTKAA (서열번호:83); (38) LAVDGSQRSA (서열번호:84); (39) LPISDQTKHA (서열번호:85); (40) LPKDATKTIA (서열번호:86); (41) LPPQDTTKNA (서열번호:87); 및 (42) PAPQDTTKNA (서열번호:88);.
양태 18. 양태 16의 rAAV 비리온으로서, 상기 펩타이드는 하기 아미노산 서열 중 하나를 포함한다: (1) LAKDATKNA (서열번호:47); (2) PAHQDTTKNA (서열번호:48); (3) LAHQDTTKNA (서열번호:49); (4) LATTSQNKPA (서열번호:50); (5) LAISDQTKHA (서열번호:51); (6) IARGVAPSSA (서열번호:52); (7) LAPDSTTRSA (서열번호:53); (8) LAKGTELKPA (서열번호:54); (9) LAIIDATKNA (서열번호:55); (10) LAVDGAQRSA (서열번호:56); (11) PAPQDTTKKA (서열번호:57); (12) LPHQDTTKNA (서열번호:58); (13) LAKDATKTIA (서열번호:59); (14) LAKQQSASTA (서열번호:60); (15) LAKSDQSKPA (서열번호:61); (16) LSHQDTTKNA (서열번호:62); (17) LAANQPSKPA (서열번호:63); 및 (18) LAVSDSTKAA (서열번호:64).
양태 19. 하기를 포함하는 약제학적 조성물: a) 양태 1 내지 18 중 임의의 하나의 재조합 아데노-관련된 바이러스 비리온; 및 b) 약제학적으로 허용가능한 부형제.
양태 20. 개체에서 유전자 산물을 망막 세포에 전달하는 방법으로서, 상기 방법은 상기 개체에게 양태 1 내지 18 중 임의의 하나에 따른 재조합 아데노-관련된 바이러스 (rAAV) 비리온을 투여하는 것을 포함한다.
양태 21. 양태 20의 방법으로서, 상기 유전자 산물은 폴리펩타이드이다.
양태 22. 양태 20의 방법으로서, 상기 유전자 산물은 짧은 간섭 RNA 또는 압타머이다.
양태 23. 양태 21의 방법으로서, 상기 폴리펩타이드는 신경보호성 인자, 항-혈관신생 폴리펩타이드, 항-세포자멸적 인자, 또는 망막 세포의 기능을 향상시키는 폴리펩타이드이다.
양태 24. 양태 21의 방법으로서, 상기 폴리펩타이드는 신경교 유래된 신경친화성 인자, 섬유모세포 성장 인자 2, 뉴르투린, 섬모 신경친화성 인자, 신경 성장 인자, 뇌 유래된 신경친화성 인자, 표피 성장 인자, 로돕신, 세포자멸사의 X-연결된 억제제, 레티노쉬신, RPE65, 색소성 망막염 GTPase-상호작용 단백질-1, 페리페린, 페리페린-2, 로돕신, RdCVF, 색소성 망막염 GTPase 조절자 (RPGR), 또는 소닉 헤지혹이다.
양태 25. 양태 21의 방법으로서, 상기 폴리펩타이드는 RNA-유도된 엔도뉴클레아제이다.
양태 26. 안구 질환을 치료하는 방법으로서, 상기 방법은 그것이 필요한 개체에게 양태 1 내지 18 중 임의의 하나에 따른 재조합 아데노-관련된 바이러스 (rAAV) 비리온의 유효량을 투여하는 것을 포함한다.
양태 27. 양태 26의 방법으로서, 상기 투여는 안구내 주사에 의한 것이다.
양태 28. 양태 26의 방법으로서, 상기 투여는 유리체내 주사에 의한 것이다.
양태 29. 양태 26의 방법으로서, 상기 안구 질환은 녹내장, 색소성 망막염, 황반 변성, 망막층간분리, 레버 선천성 흑내장, 당뇨 망막병증, 색맹, 또는 색상 시각상실이다.
양태 30. 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질을 인코딩하는 뉴클레오타이드 서열을 포함하는 단리된 핵산으로서, 상기 변이체 AAV 캡시드 단백질은 상응하는 친계 AAV 캡시드 단백질에 대해 캡시드 단백질 GH 루프에서 약 5개의 아미노산 내지 약 20개의 아미노산의 삽입을 포함하고, 그리고 상기 변이체 캡시드 단백질은, AAV 비리온 내에서 존재할 때, 망막 세포의 AAV 비리온의 증가된 감염성을 제공하고, 그리고 상기 아미노산 삽입은 천연 AAV 캡시드의 GH 루프에 있고, 상기 삽입은 식 I-X 중 임의의 것의 펩타이드이다.
양태 31. 양태 30의 단리된 핵산으로서, 삽입 부위는 AAV2의 아미노산 587 내지 588, AAV1의 아미노산 590 내지 591, AAV5의 아미노산 575 내지 576, AAV6의 아미노산 590 내지 591, AAV7의 아미노산 589 내지 590, AAV8의 아미노산 590 내지 591, AAV9의 아미노산 588 내지 589, 또는 AAV10의 아미노산 588 내지 589이다.
양태 32. 양태 30 또는 양태 31의 핵산을 포함하는, 단리된, 유전자 변형된 숙주세포.
양태 33. 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질로서, 상기 변이체 AAV 캡시드 단백질은 약 5개의 아미노산 내지 약 20개의 아미노산의 삽입을 포함하고, 상기 아미노산 삽입은 천연 AAV 캡시드의 GH 루프에 있고, 상기 삽입은 식 I-X 중 임의의 것의 펩타이드이다.
양태 34. 양태 1 내지 33 중 임의의 것에서, 상기 GH 루프에 삽입된 이종성 펩타이드 식 I-X 중 하나일 수 있다,
식 I은 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu, Ile, Pro, 또는 Gln이고;
X2는 Ala, Pro, Ser, Asp, Gly, Thr, 또는 Val이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, 또는 Tyr이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, 또는 Arg이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, 또는 Tyr이고;
X6는 Thr, Ala, Gln, Ser, Glu, Pro, 또는 Ile;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, 또는 Cys이고;
X8는 Lys, Ser, Arg, Thr, Ala, Glu, Ile, 또는 Asn이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Phe, Asp, Thr, Val, 또는 Met이고;
식 II는 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala이고;
식 III는 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Thr, Asp Val, 또는 Met이고;
식 IV는 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu이고;
X2는 Ala이고;
X3는 Lys, His, Thr, Ile, Pro, 또는 Val이고;
X4 (존재하면)는 Gln, Asp, Ser, 또는 Gly이고;
X5는 Asp, Ser, 또는 Gln이고;
X6는 Thr, Ala, Gln, 또는 Ser이고;
X7는 Thr 또는 Ser이고;
X8는 Lys, Ser, 또는 Arg이고;
X9는 Asn, Pro, 또는 Ser이고; 그리고
X10는 Ala이고;
식 V는 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu이고;
X2는 Ala이고;
X3는 Lys 또는 His이고;
X4 (존재하면)는 Gln, Asp, Ser, 또는 Gly이고;
X5는 Asp, Ser, 또는 Gln이고;
X6는 Thr, Ala, Gln, 또는 Ser이고;
X7는 Thr 또는 Ser이고;
X8는 Lys, Ser, 또는 Arg이고;
X9는 Asn, Pro, 또는 Ser이고; 그리고
X10는 Ala이고;
식 VI은 X1X2X3X4X5X6X7X8X9X10이되,
X1는 Leu이고;
X2는 Ala이고;
X3는 Asn, Lys, Thr, Gln, Ser, Ile, 또는 Leu이고;
X4는 Ser, Ala, Thr, Glu, Gln, Gly, Lys, 또는 Pro이고;
X5는 Asp, Pro, Glu, Thr, Asn, 또는 Arg이고;
X6는 Ile, His, Thr, Gln, Asn, Tyr, Asp, 또는 Glu이고;
X7는 Gln, Thr, Asn, Ala, 또는 Lys이고;
X8는 Lys, Thr, Arg, 또는 Asp이고;
X9는 Pro, Asn, Thr, Arg, Lys, 또는 Ser이고; 그리고
X10는 Ala이고;
식 VII는 LAHQDTTKX1X2X3 (서열번호:148)이되, 상기 X1는 Lys, Thr, Asn, 또는 His이고; X2는 Ala, Thr, Val, Ile, Met, 또는 Asp이고; 그리고 X3는, 존재한다면, Ala이고;
식 VIII는 LAX1QX2TX3X4X5X6 (서열번호:149)이되, 상기 X1는 Ala, Pro, Asp, 또는 His이고; X2는 Gly 또는 Asp이고; X3는 Ala, Thr, 또는 Lys이고; X4는 Asn, Glu, Lys, Arg, 또는 Thr이고; X5는 Leu, Asn, Lys, 또는 Thr이고; 그리고 X6는, 존재한다면, Ala, Thr, Asp, Val, 또는 Met이고;
식 IX는 X1AX2X3DX4TKX5A (서열번호:150)이되, 상기 X1는 Val 또는 Leu이고; X2는 Ile, Val, His, 또는 Asp이고; X3는 Glu, Ser, Lys, 또는 Gln이고; X4는 His, Ser, 또는 Thr이고; 그리고 X5는 Ser, Ala, Asn, His, 또는 Lys이고; 그리고
식 X는 X1X2X3AX4QX5TX6KNA (서열번호:151)이되, 상기 X1는, 존재한다면, Leu이고; X2는, 존재한다면, Ala이고; X3는 Lys, Leu, 또는 Pro이고; X4는 Asn, His, Pro, 또는 Tyr이고; X5는 Asn, Gly, Val, 또는 Asp이고; 그리고 X6는 Pro 또는 Thr이다.
실시예
하기 실시예는 본 발명의 제조 방법 및 사용 방법의 완전한 개시 및 기재를 종래기술의 숙련가에 제공하기 위해 제시되고, 본 발명가가 그들의 발명으로서 고려하였던 것의 범위를 제한하기 위한 의도도 아니고 이들이 아래 실험이 수행된 모든 또는 유일한 실험이라는 것을 나타내기 위한 의도도 아니다. 사용된 숫자(예를 들면, 양, 온도, 등)에 관하여 정확성을 확보하기 위해 노력하였지만 일부 실험 오차 및 편차는 고려될 수 있다. 달리 나타내지 않는 한, 부는 중량부이고, 분자량은 중량 평균 분자량이고, 온도는 섭씨 온도이고, 압력은 대기압 또는 근처이다. 표준 약어는 사용될 수 있다, 예를 들어, bp, 염기 쌍(들); kb, 킬로염기(들); pl, 피코리터(들); s 또는 sec, 초(들); min, 분(들); h 또는 hr, 시(들); aa, 아미노산(들); kb, 킬로염기(들); bp, 염기 쌍(들); nt, 뉴클레오타이드(들); i.m., 근육내(로); i.p., 복강내(로); s.c., 피하(로); 기타 동종.
실시예 1: AAV 캡시드 변이체를 가진 AAV 비리온의 일반화 및 특성화
반복 생체내 스크리닝 방법론은 대형 동물 눈에서 범망막 AAV 감염을 예방하는 중요한 그리고 복잡한 장벽을 극복할 수 있는 캡시드 변이체를 가진 AAV를 창출하는데 사용되었다. 개는, 인간과 유사한 눈 크기 및 구조를 가진, 망막 변성 질환에 대하여 중요한 전임상 모델이고, 망막 질환의 많은 형태가 다양한 개 품종에서 자연적으로 발생하고 있다. 스크리닝 방법은 개 망막에서 범망막 감염할 수 있는 96 AAV 변이체를 확인하는데 사용되었다. 심층 서열분석은 개 망막내 스크리닝된 AAV의 풀로부터 이들 변이체의 18의 성능을 정량화하는데 사용되었다. 감염성은 유리체내 주사 이후 망막 세포에서 바이러스 DNA 및 mRNA의 수준을 기준으로 하여 정량화되었다. 이들 변이체는 대형 동물 및 인간 눈에서 광범위의 유전자 전달 전략에 사용될 수 있다.
AAV 캡시드상의 표면 노출된 위치에서 (5' 6-뉴클레오타이드 링커 및 3' 3-뉴클레오타이드 링커에 의해 둘러싸인) 랜덤 21-뉴클레오타이드 삽입물을 함유하는 펩타이드 디스플레이 라이브러리는 창출되었다. 각각의 바이러스 게놈이 그 게놈이 인코딩하였던 캡시드 단백질 쉘 내에 캡시드화되었던 정도로 바이러스는 패키징되었다. 그러므로 선택을 통해 확인된 기능적 개량은 바이러스 캡시드 내에 함유된 게놈 서열에 연결될 수 있다. 이러한 라이브러리로부터, 반복 생체내 스크리닝 선택 공정은 유리체로부터 개 망막을 감염시키는 능력을 가진 변이체를 확인하는데 사용되었다 (도 1). 개 눈은 ~250 μL의 10E+13 - 10E+14 바이러스 게놈/mL (vg/mL) 역가 바이러스로 각각의 라운드에서 주사되었다. 주사 3주 후, 눈은 적출되었고, 망막 펀치는 망막의 중심 및 주변 영역으로부터 실시되었다. RPE 세포는 망막 조직으로부터 분리되었고, 조직은 냉동되었다. DNA는 그 다음 망막 세포로부터 수집되었고, cap 유전자는 단리된 샘플로부터 폴리머라제 사슬 반응(PCR) 증폭되었다. Cap 유전자는 후속 AAV 패키징에 사용되었다.
도 1. 개 망막 AAV 변이체를 개발하는데 사용된 지향성 진화 방법론의 예시. 펩타이드 디스플레이 라이브러리는 창출되었고, AAV 벡터에 패키징되었고, 유리체내 주사를 통해 개 눈에 주사되었다. 선택의 반복 라운드는 벡터의 풀로부터 AAV 변이체를 양성적으로 선택하는데 사용되었다. 선택의 3 라운드는 오류 발생이 쉬운 PCR의 라운드가 뒤따랐고, 추가의 선택 라운드가 뒤따랐다.
선택의 5 라운드 이후, 일루미나 심층 서열분석은 AAV 변이체의 라이브러리에서 관련 표시에서 라운드에 걸쳐 증가하였던 변이체를 확인하는데 사용되었다. 바이러스 라이브러리에서 표시의 증가는 유리체로부터 개 망막을 감염시키는 능력 및 양성 선택을 나타낸다. ~10E+7 변이체의 라이브러리 중에서, 생체내 스크린에서 선택되었던 상위 96 변이체는 표 1에서 제공된다.
표 1에서 묘사된 96 변이체 중 상위 18 변이체의 개 망막을 감염시키는 능력은 고 처리 서열분석을 사용하여 추가로 정량화되었다. 표 2는 추가 정량화를 위하여 선택된 상위 18 변이체를 묘사한다.
18 변이체는 GFP의 유비쿼터스 CAG 프로모터 구동 발현으로 패키징되었다. GFP cDNA는 고유 25 염기-쌍 바 코드 식별기에 융합되었다. 각각의 18 변이체는 고유 GFP 바코드로 패키징되었다. 패키징된 변이체는 동일 비율로 혼합되었고, 대조군 AAV2-기반 벡터 (자연적으로 발생하는 친계 혈청형을 나타내는 음성 대조군)과 함께, 망막 속에 주사되었다. 주사 후, DNA 및 mRNA는 광수용체 및 RPE 세포로부터 수집되었다. DNA 및 mRNA 수준은 망막에 DNA를 전달하기 위한 그리고 전이유전자 발현을 초래하기 위한 개-유도된 벡터의 능력을 결정하기 위해 정량화되었다 (도 2).
도 2. GFP-바코드 작제물을 함유하는 변이체의 심층 서열분석. 개-유도된 변이체에 의한 개 망막의 감염은 태깅된 GFP cDNA 및 mRNA의 심층 서열분석에 의해 정량화되었다.
18-구성원 라이브러리의 발현은 냉동된 망막 부문의 공초점 현미경을 사용하여 이미지화되었다. GFP 발현은 내부 망막에서 망막 세포, 그리고 외부 망막에서 광수용체가 18-구성원 라이브러리로 표적화되었다는 것을 보여주었다 (도 3).
도 3. 18-구성원 개-유도된 AAV 변이체 라이브러리는 결절종 세포층, 내부 핵층, 광수용체 층, 및 RPE 층에서 세포를 감염시킨다.
시험된 상위 18 변이체 중에서, 2 변이체는 최고 수준의 DNA 및 mRNA 회수를 초래하였다. 최고 수준의 DNA 회수를 초래하는 변이체는 삽입 서열 ~588- PAPQDTTKKA (서열번호:57) 을 가졌다. 최고 수준의 mRNA 발현을 초래하는 변이체는 삽입 서열 ~588- LAPDSTTRSA (서열번호:53)을 가졌다.
본 발명이 이의 특정 구현예를 참조로 기재되어 있는 동안, 본 발명의 진정한 사상 및 범위에서 이탈 없이 다양한 변화가 실시될 수 있고 등가물이 치환될 수 있다는 것이 종래기술의 숙련가에 의해 이해되어야 한다. 게다가, 특정한 상황, 물질, 당해 조성물, 공정, 공정 단계 또는 단계들을, 본 발명의 목적, 사상 및 범위에 적응시키기 위해 다수 변형이 실시될 수 있다. 모든 그와 같은 변형은 여기에 첨부된 청구항의 범위 내이도록 의도된다.
<110> The Regents of the University of California
<120> ADENO-ASSOCIATED VIRUS VIRIONS WITH VARIANT CAPSID AND METHODS OF
USE THEREOF
<130> BERK-335WO
<150> 62/368,929
<151> 2016-07-29
<160> 152
<170> KoPatentIn version 3.0
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<213> Adeno-associated virus-2
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<212> PRT
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Leu Ser Lys Phe Gln Asp Ser Ser Gln Trp Leu Gln Ile Asp Leu Lys
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Glu Ile Lys Val Ile Ser Gly Ile Leu Thr Gln Gly Arg Cys Asp Ile
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Leu Asn Trp Ile Tyr Tyr Lys Asp Gln Thr Gly Asn Asn Arg Val Phe
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Tyr Lys Asn Tyr Leu Asp Ala Ala Asn Met Ser Met Met Val Leu Arg
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His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser Ile
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Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Gly Gln
165 170 175
Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr
180 185 190
Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln Cys
195 200 205
Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys
210 215 220
Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Thr
225 230 235 240
Leu Thr Ile Lys Arg Gly Arg
245
<210> 12
<211> 533
<212> PRT
<213> Homo sapiens
<400> 12
Met Ser Ile Gln Val Glu His Pro Ala Gly Gly Tyr Lys Lys Leu Phe
1 5 10 15
Glu Thr Val Glu Glu Leu Ser Ser Pro Leu Thr Ala His Val Thr Gly
20 25 30
Arg Ile Pro Leu Trp Leu Thr Gly Ser Leu Leu Arg Cys Gly Pro Gly
35 40 45
Leu Phe Glu Val Gly Ser Glu Pro Phe Tyr His Leu Phe Asp Gly Gln
50 55 60
Ala Leu Leu His Lys Phe Asp Phe Lys Glu Gly His Val Thr Tyr His
65 70 75 80
Arg Arg Phe Ile Arg Thr Asp Ala Tyr Val Arg Ala Met Thr Glu Lys
85 90 95
Arg Ile Val Ile Thr Glu Phe Gly Thr Cys Ala Phe Pro Asp Pro Cys
100 105 110
Lys Asn Ile Phe Ser Arg Phe Phe Ser Tyr Phe Arg Gly Val Glu Val
115 120 125
Thr Asp Asn Ala Leu Val Asn Val Tyr Pro Val Gly Glu Asp Tyr Tyr
130 135 140
Ala Cys Thr Glu Thr Asn Phe Ile Thr Lys Ile Asn Pro Glu Thr Leu
145 150 155 160
Glu Thr Ile Lys Gln Val Asp Leu Cys Asn Tyr Val Ser Val Asn Gly
165 170 175
Ala Thr Ala His Pro His Ile Glu Asn Asp Gly Thr Val Tyr Asn Ile
180 185 190
Gly Asn Cys Phe Gly Lys Asn Phe Ser Ile Ala Tyr Asn Ile Val Lys
195 200 205
Ile Pro Pro Leu Gln Ala Asp Lys Glu Asp Pro Ile Ser Lys Ser Glu
210 215 220
Ile Val Val Gln Phe Pro Cys Ser Asp Arg Phe Lys Pro Ser Tyr Val
225 230 235 240
His Ser Phe Gly Leu Thr Pro Asn Tyr Ile Val Phe Val Glu Thr Pro
245 250 255
Val Lys Ile Asn Leu Phe Lys Phe Leu Ser Ser Trp Ser Leu Trp Gly
260 265 270
Ala Asn Tyr Met Asp Cys Phe Glu Ser Asn Glu Thr Met Gly Val Trp
275 280 285
Leu His Ile Ala Asp Lys Lys Arg Lys Lys Tyr Leu Asn Asn Lys Tyr
290 295 300
Arg Thr Ser Pro Phe Asn Leu Phe His His Ile Asn Thr Tyr Glu Asp
305 310 315 320
Asn Gly Phe Leu Ile Val Asp Leu Cys Cys Trp Lys Gly Phe Glu Phe
325 330 335
Val Tyr Asn Tyr Leu Tyr Leu Ala Asn Leu Arg Glu Asn Trp Glu Glu
340 345 350
Val Lys Lys Asn Ala Arg Lys Ala Pro Gln Pro Glu Val Arg Arg Tyr
355 360 365
Val Leu Pro Leu Asn Ile Asp Lys Ala Asp Thr Gly Lys Asn Leu Val
370 375 380
Thr Leu Pro Asn Thr Thr Ala Thr Ala Ile Leu Cys Ser Asp Glu Thr
385 390 395 400
Ile Trp Leu Glu Pro Glu Val Leu Phe Ser Gly Pro Arg Gln Ala Phe
405 410 415
Glu Phe Pro Gln Ile Asn Tyr Gln Lys Tyr Cys Gly Lys Pro Tyr Thr
420 425 430
Tyr Ala Tyr Gly Leu Gly Leu Asn His Phe Val Pro Asp Arg Leu Cys
435 440 445
Lys Leu Asn Val Lys Thr Lys Glu Thr Trp Val Trp Gln Glu Pro Asp
450 455 460
Ser Tyr Pro Ser Glu Pro Ile Phe Val Ser His Pro Asp Ala Leu Glu
465 470 475 480
Glu Asp Asp Gly Val Val Leu Ser Val Val Val Ser Pro Gly Ala Gly
485 490 495
Gln Lys Pro Ala Tyr Leu Leu Ile Leu Asn Ala Lys Asp Leu Ser Glu
500 505 510
Val Ala Arg Ala Glu Val Glu Ile Asn Ile Pro Val Thr Phe His Gly
515 520 525
Leu Phe Lys Lys Ser
530
<210> 13
<211> 346
<212> PRT
<213> Homo sapiens
<400> 13
Met Ala Leu Leu Lys Val Lys Phe Asp Gln Lys Lys Arg Val Lys Leu
1 5 10 15
Ala Gln Gly Leu Trp Leu Met Asn Trp Phe Ser Val Leu Ala Gly Ile
20 25 30
Ile Ile Phe Ser Leu Gly Leu Phe Leu Lys Ile Glu Leu Arg Lys Arg
35 40 45
Ser Asp Val Met Asn Asn Ser Glu Ser His Phe Val Pro Asn Ser Leu
50 55 60
Ile Gly Met Gly Val Leu Ser Cys Val Phe Asn Ser Leu Ala Gly Lys
65 70 75 80
Ile Cys Tyr Asp Ala Leu Asp Pro Ala Lys Tyr Ala Arg Trp Lys Pro
85 90 95
Trp Leu Lys Pro Tyr Leu Ala Ile Cys Val Leu Phe Asn Ile Ile Leu
100 105 110
Phe Leu Val Ala Leu Cys Cys Phe Leu Leu Arg Gly Ser Leu Glu Asn
115 120 125
Thr Leu Gly Gln Gly Leu Lys Asn Gly Met Lys Tyr Tyr Arg Asp Thr
130 135 140
Asp Thr Pro Gly Arg Cys Phe Met Lys Lys Thr Ile Asp Met Leu Gln
145 150 155 160
Ile Glu Phe Lys Cys Cys Gly Asn Asn Gly Phe Arg Asp Trp Phe Glu
165 170 175
Ile Gln Trp Ile Ser Asn Arg Tyr Leu Asp Phe Ser Ser Lys Glu Val
180 185 190
Lys Asp Arg Ile Lys Ser Asn Val Asp Gly Arg Tyr Leu Val Asp Gly
195 200 205
Val Pro Phe Ser Cys Cys Asn Pro Ser Ser Pro Arg Pro Cys Ile Gln
210 215 220
Tyr Gln Ile Thr Asn Asn Ser Ala His Tyr Ser Tyr Asp His Gln Thr
225 230 235 240
Glu Glu Leu Asn Leu Trp Val Arg Gly Cys Arg Ala Ala Leu Leu Ser
245 250 255
Tyr Tyr Ser Ser Leu Met Asn Ser Met Gly Val Val Thr Leu Leu Ile
260 265 270
Trp Leu Phe Glu Val Thr Ile Thr Ile Gly Leu Arg Tyr Leu Gln Thr
275 280 285
Ser Leu Asp Gly Val Ser Asn Pro Glu Glu Ser Glu Ser Glu Ser Gln
290 295 300
Gly Trp Leu Leu Glu Arg Ser Val Pro Glu Thr Trp Lys Ala Phe Leu
305 310 315 320
Glu Ser Val Lys Lys Leu Gly Lys Gly Asn Gln Val Glu Ala Glu Gly
325 330 335
Ala Asp Ala Gly Gln Ala Pro Glu Ala Gly
340 345
<210> 14
<211> 470
<212> PRT
<213> Homo sapiens
<400> 14
Met Ser His His Pro Ser Gly Leu Arg Ala Gly Phe Ser Ser Thr Ser
1 5 10 15
Tyr Arg Arg Thr Phe Gly Pro Pro Pro Ser Leu Ser Pro Gly Ala Phe
20 25 30
Ser Tyr Ser Ser Ser Ser Arg Phe Ser Ser Ser Arg Leu Leu Gly Ser
35 40 45
Ala Ser Pro Ser Ser Ser Val Arg Leu Gly Ser Phe Arg Ser Pro Arg
50 55 60
Ala Gly Ala Gly Ala Leu Leu Arg Leu Pro Ser Glu Arg Leu Asp Phe
65 70 75 80
Ser Met Ala Glu Ala Leu Asn Gln Glu Phe Leu Ala Thr Arg Ser Asn
85 90 95
Glu Lys Gln Glu Leu Gln Glu Leu Asn Asp Arg Phe Ala Asn Phe Ile
100 105 110
Glu Lys Val Arg Phe Leu Glu Gln Gln Asn Ala Ala Leu Arg Gly Glu
115 120 125
Leu Ser Gln Ala Arg Gly Gln Glu Pro Ala Arg Ala Asp Gln Leu Cys
130 135 140
Gln Gln Glu Leu Arg Glu Leu Arg Arg Glu Leu Glu Leu Leu Gly Arg
145 150 155 160
Glu Arg Asp Arg Val Gln Val Glu Arg Asp Gly Leu Ala Glu Asp Leu
165 170 175
Ala Ala Leu Lys Gln Arg Leu Glu Glu Glu Thr Arg Lys Arg Glu Asp
180 185 190
Ala Glu His Asn Leu Val Leu Phe Arg Lys Asp Val Asp Asp Ala Thr
195 200 205
Leu Ser Arg Leu Glu Leu Glu Arg Lys Ile Glu Ser Leu Met Asp Glu
210 215 220
Ile Glu Phe Leu Lys Lys Leu His Glu Glu Glu Leu Arg Asp Leu Gln
225 230 235 240
Val Ser Val Glu Ser Gln Gln Val Gln Gln Val Glu Val Glu Ala Thr
245 250 255
Val Lys Pro Glu Leu Thr Ala Ala Leu Arg Asp Ile Arg Ala Gln Tyr
260 265 270
Glu Ser Ile Ala Ala Lys Asn Leu Gln Glu Ala Glu Glu Trp Tyr Lys
275 280 285
Ser Lys Tyr Ala Asp Leu Ser Asp Ala Ala Asn Arg Asn His Glu Ala
290 295 300
Leu Arg Gln Ala Lys Gln Glu Met Asn Glu Ser Arg Arg Gln Ile Gln
305 310 315 320
Ser Leu Thr Cys Glu Val Asp Gly Leu Arg Gly Thr Asn Glu Ala Leu
325 330 335
Leu Arg Gln Leu Arg Glu Leu Glu Glu Gln Phe Ala Leu Glu Ala Gly
340 345 350
Gly Tyr Gln Ala Gly Ala Ala Arg Leu Glu Glu Glu Leu Arg Gln Leu
355 360 365
Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln Glu Leu Leu Asn
370 375 380
Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu
385 390 395 400
Glu Gly Glu Glu Ser Arg Ile Ser Val Pro Val His Ser Phe Ala Ser
405 410 415
Leu Asn Ile Lys Thr Thr Val Pro Glu Val Glu Pro Pro Gln Asp Ser
420 425 430
His Ser Arg Lys Thr Val Leu Ile Lys Thr Ile Glu Thr Arg Asn Gly
435 440 445
Glu Val Val Thr Glu Ser Gln Lys Glu Gln Arg Ser Glu Leu Asp Lys
450 455 460
Ser Ser Ala His Ser Tyr
465 470
<210> 15
<211> 1286
<212> PRT
<213> Homo sapiens
<400> 15
Met Ser His Leu Val Asp Pro Thr Ser Gly Asp Leu Pro Val Arg Asp
1 5 10 15
Ile Asp Ala Ile Pro Leu Val Leu Pro Ala Ser Lys Gly Lys Asn Met
20 25 30
Lys Thr Gln Pro Pro Leu Ser Arg Met Asn Arg Glu Glu Leu Glu Asp
35 40 45
Ser Phe Phe Arg Leu Arg Glu Asp His Met Leu Val Lys Glu Leu Ser
50 55 60
Trp Lys Gln Gln Asp Glu Ile Lys Arg Leu Arg Thr Thr Leu Leu Arg
65 70 75 80
Leu Thr Ala Ala Gly Arg Asp Leu Arg Val Ala Glu Glu Ala Ala Pro
85 90 95
Leu Ser Glu Thr Ala Arg Arg Gly Gln Lys Ala Gly Trp Arg Gln Arg
100 105 110
Leu Ser Met His Gln Arg Pro Gln Met His Arg Leu Gln Gly His Phe
115 120 125
His Cys Val Gly Pro Ala Ser Pro Arg Arg Ala Gln Pro Arg Val Gln
130 135 140
Val Gly His Arg Gln Leu His Thr Ala Gly Ala Pro Val Pro Glu Lys
145 150 155 160
Pro Lys Arg Gly Pro Arg Asp Arg Leu Ser Tyr Thr Ala Pro Pro Ser
165 170 175
Phe Lys Glu His Ala Thr Asn Glu Asn Arg Gly Glu Val Ala Ser Lys
180 185 190
Pro Ser Glu Leu Val Ser Gly Ser Asn Ser Ile Ile Ser Phe Ser Ser
195 200 205
Val Ile Ser Met Ala Lys Pro Ile Gly Leu Cys Met Pro Asn Ser Ala
210 215 220
His Ile Met Ala Ser Asn Thr Met Gln Val Glu Glu Pro Pro Lys Ser
225 230 235 240
Pro Glu Lys Met Trp Pro Lys Asp Glu Asn Phe Glu Gln Arg Ser Ser
245 250 255
Leu Glu Cys Ala Gln Lys Ala Ala Glu Leu Arg Ala Ser Ile Lys Glu
260 265 270
Lys Val Glu Leu Ile Arg Leu Lys Lys Leu Leu His Glu Arg Asn Ala
275 280 285
Ser Leu Val Met Thr Lys Ala Gln Leu Thr Glu Val Gln Glu Ala Tyr
290 295 300
Glu Thr Leu Leu Gln Lys Asn Gln Gly Ile Leu Ser Ala Ala His Glu
305 310 315 320
Ala Leu Leu Lys Gln Val Asn Glu Leu Arg Ala Glu Leu Lys Glu Glu
325 330 335
Ser Lys Lys Ala Val Ser Leu Lys Ser Gln Leu Glu Asp Val Ser Ile
340 345 350
Leu Gln Met Thr Leu Lys Glu Phe Gln Glu Arg Val Glu Asp Leu Glu
355 360 365
Lys Glu Arg Lys Leu Leu Asn Asp Asn Tyr Asp Lys Leu Leu Glu Ser
370 375 380
Met Leu Asp Ser Ser Asp Ser Ser Ser Gln Pro His Trp Ser Asn Glu
385 390 395 400
Leu Ile Ala Glu Gln Leu Gln Gln Gln Val Ser Gln Leu Gln Asp Gln
405 410 415
Leu Asp Ala Glu Leu Glu Asp Lys Arg Lys Val Leu Leu Glu Leu Ser
420 425 430
Arg Glu Lys Ala Gln Asn Glu Asp Leu Lys Leu Glu Val Thr Asn Ile
435 440 445
Leu Gln Lys His Lys Gln Glu Val Glu Leu Leu Gln Asn Ala Ala Thr
450 455 460
Ile Ser Gln Pro Pro Asp Arg Gln Ser Glu Pro Ala Thr His Pro Ala
465 470 475 480
Val Leu Gln Glu Asn Thr Gln Ile Glu Pro Ser Glu Pro Lys Asn Gln
485 490 495
Glu Glu Lys Lys Leu Ser Gln Val Leu Asn Glu Leu Gln Val Ser His
500 505 510
Ala Glu Thr Thr Leu Glu Leu Glu Lys Thr Arg Asp Met Leu Ile Leu
515 520 525
Gln Arg Lys Ile Asn Val Cys Tyr Gln Glu Glu Leu Glu Ala Met Met
530 535 540
Thr Lys Ala Asp Asn Asp Asn Arg Asp His Lys Glu Lys Leu Glu Arg
545 550 555 560
Leu Thr Arg Leu Leu Asp Leu Lys Asn Asn Arg Ile Lys Gln Leu Glu
565 570 575
Gly Ile Leu Arg Ser His Asp Leu Pro Thr Ser Glu Gln Leu Lys Asp
580 585 590
Val Ala Tyr Gly Thr Arg Pro Leu Ser Leu Cys Leu Glu Thr Leu Pro
595 600 605
Ala His Gly Asp Glu Asp Lys Val Asp Ile Ser Leu Leu His Gln Gly
610 615 620
Glu Asn Leu Phe Glu Leu His Ile His Gln Ala Phe Leu Thr Ser Ala
625 630 635 640
Ala Leu Ala Gln Ala Gly Asp Thr Gln Pro Thr Thr Phe Cys Thr Tyr
645 650 655
Ser Phe Tyr Asp Phe Glu Thr His Cys Thr Pro Leu Ser Val Gly Pro
660 665 670
Gln Pro Leu Tyr Asp Phe Thr Ser Gln Tyr Val Met Glu Thr Asp Ser
675 680 685
Leu Phe Leu His Tyr Leu Gln Glu Ala Ser Ala Arg Leu Asp Ile His
690 695 700
Gln Ala Met Ala Ser Glu His Ser Thr Leu Ala Ala Gly Trp Ile Cys
705 710 715 720
Phe Asp Arg Val Leu Glu Thr Val Glu Lys Val His Gly Leu Ala Thr
725 730 735
Leu Ile Gly Ala Gly Gly Glu Glu Phe Gly Val Leu Glu Tyr Trp Met
740 745 750
Arg Leu Arg Phe Pro Ile Lys Pro Ser Leu Gln Ala Cys Asn Lys Arg
755 760 765
Lys Lys Ala Gln Val Tyr Leu Ser Thr Asp Val Leu Gly Gly Arg Lys
770 775 780
Ala Gln Glu Glu Glu Phe Arg Ser Glu Ser Trp Glu Pro Gln Asn Glu
785 790 795 800
Leu Trp Ile Glu Ile Thr Lys Cys Cys Gly Leu Arg Ser Arg Trp Leu
805 810 815
Gly Thr Gln Pro Ser Pro Tyr Ala Val Tyr Arg Phe Phe Thr Phe Ser
820 825 830
Asp His Asp Thr Ala Ile Ile Pro Ala Ser Asn Asn Pro Tyr Phe Arg
835 840 845
Asp Gln Ala Arg Phe Pro Val Leu Val Thr Ser Asp Leu Asp His Tyr
850 855 860
Leu Arg Arg Glu Ala Leu Ser Ile His Val Phe Asp Asp Glu Asp Leu
865 870 875 880
Glu Pro Gly Ser Tyr Leu Gly Arg Ala Arg Val Pro Leu Leu Pro Leu
885 890 895
Ala Lys Asn Glu Ser Ile Lys Gly Asp Phe Asn Leu Thr Asp Pro Ala
900 905 910
Glu Lys Pro Asn Gly Ser Ile Gln Val Gln Leu Asp Trp Lys Phe Pro
915 920 925
Tyr Ile Pro Pro Glu Ser Phe Leu Lys Pro Glu Ala Gln Thr Lys Gly
930 935 940
Lys Asp Thr Lys Asp Ser Ser Lys Ile Ser Ser Glu Glu Glu Lys Ala
945 950 955 960
Ser Phe Pro Ser Gln Asp Gln Met Ala Ser Pro Glu Val Pro Ile Glu
965 970 975
Ala Gly Gln Tyr Arg Ser Lys Arg Lys Pro Pro His Gly Gly Glu Arg
980 985 990
Lys Glu Lys Glu His Gln Val Val Ser Tyr Ser Arg Arg Lys His Gly
995 1000 1005
Lys Arg Ile Gly Val Gln Gly Lys Asn Arg Met Glu Tyr Leu Ser Leu
1010 1015 1020
Asn Ile Leu Asn Gly Asn Thr Pro Glu Gln Val Asn Tyr Thr Glu Trp
1025 1030 1035 1040
Lys Phe Ser Glu Thr Asn Ser Phe Ile Gly Asp Gly Phe Lys Asn Gln
1045 1050 1055
His Glu Glu Glu Glu Met Thr Leu Ser His Ser Ala Leu Lys Gln Lys
1060 1065 1070
Glu Pro Leu His Pro Val Asn Asp Lys Glu Ser Ser Glu Gln Gly Ser
1075 1080 1085
Glu Val Ser Glu Ala Gln Thr Thr Asp Ser Asp Asp Val Ile Val Pro
1090 1095 1100
Pro Met Ser Gln Lys Tyr Pro Lys Ala Asp Ser Glu Lys Met Cys Ile
1105 1110 1115 1120
Glu Ile Val Ser Leu Ala Phe Tyr Pro Glu Ala Glu Val Met Ser Asp
1125 1130 1135
Glu Asn Ile Lys Gln Val Tyr Val Glu Tyr Lys Phe Tyr Asp Leu Pro
1140 1145 1150
Leu Ser Glu Thr Glu Thr Pro Val Ser Leu Arg Lys Pro Arg Ala Gly
1155 1160 1165
Glu Glu Ile His Phe His Phe Ser Lys Val Ile Asp Leu Asp Pro Gln
1170 1175 1180
Glu Gln Gln Gly Arg Arg Arg Phe Leu Phe Asp Met Leu Asn Gly Gln
1185 1190 1195 1200
Asp Pro Asp Gln Gly His Leu Lys Phe Thr Val Val Ser Asp Pro Leu
1205 1210 1215
Asp Glu Glu Lys Lys Glu Cys Glu Glu Val Gly Tyr Ala Tyr Leu Gln
1220 1225 1230
Leu Trp Gln Ile Leu Glu Ser Gly Arg Asp Ile Leu Glu Gln Glu Leu
1235 1240 1245
Asp Ile Val Ser Pro Glu Asp Leu Ala Thr Pro Ile Gly Arg Leu Lys
1250 1255 1260
Val Ser Leu Gln Ala Ala Ala Val Leu His Ala Ile Tyr Lys Glu Met
1265 1270 1275 1280
Thr Glu Asp Leu Phe Ser
1285
<210> 16
<211> 653
<212> PRT
<213> Homo sapiens
<400> 16
Met Ala Asp Thr Leu Pro Ser Glu Phe Asp Val Ile Val Ile Gly Thr
1 5 10 15
Gly Leu Pro Glu Ser Ile Ile Ala Ala Ala Cys Ser Arg Ser Gly Arg
20 25 30
Arg Val Leu His Val Asp Ser Arg Ser Tyr Tyr Gly Gly Asn Trp Ala
35 40 45
Ser Phe Ser Phe Ser Gly Leu Leu Ser Trp Leu Lys Glu Tyr Gln Glu
50 55 60
Asn Ser Asp Ile Val Ser Asp Ser Pro Val Trp Gln Asp Gln Ile Leu
65 70 75 80
Glu Asn Glu Glu Ala Ile Ala Leu Ser Arg Lys Asp Lys Thr Ile Gln
85 90 95
His Val Glu Val Phe Cys Tyr Ala Ser Gln Asp Leu His Glu Asp Val
100 105 110
Glu Glu Ala Gly Ala Leu Gln Lys Asn His Ala Leu Val Thr Ser Ala
115 120 125
Asn Ser Thr Glu Ala Ala Asp Ser Ala Phe Leu Pro Thr Glu Asp Glu
130 135 140
Ser Leu Ser Thr Met Ser Cys Glu Met Leu Thr Glu Gln Thr Pro Ser
145 150 155 160
Ser Asp Pro Glu Asn Ala Leu Glu Val Asn Gly Ala Glu Val Thr Gly
165 170 175
Glu Lys Glu Asn His Cys Asp Asp Lys Thr Cys Val Pro Ser Thr Ser
180 185 190
Ala Glu Asp Met Ser Glu Asn Val Pro Ile Ala Glu Asp Thr Thr Glu
195 200 205
Gln Pro Lys Lys Asn Arg Ile Thr Tyr Ser Gln Ile Ile Lys Glu Gly
210 215 220
Arg Arg Phe Asn Ile Asp Leu Val Ser Lys Leu Leu Tyr Ser Arg Gly
225 230 235 240
Leu Leu Ile Asp Leu Leu Ile Lys Ser Asn Val Ser Arg Tyr Ala Glu
245 250 255
Phe Lys Asn Ile Thr Arg Ile Leu Ala Phe Arg Glu Gly Arg Val Glu
260 265 270
Gln Val Pro Cys Ser Arg Ala Asp Val Phe Asn Ser Lys Gln Leu Thr
275 280 285
Met Val Glu Lys Arg Met Leu Met Lys Phe Leu Thr Phe Cys Met Glu
290 295 300
Tyr Glu Lys Tyr Pro Asp Glu Tyr Lys Gly Tyr Glu Glu Ile Thr Phe
305 310 315 320
Tyr Glu Tyr Leu Lys Thr Gln Lys Leu Thr Pro Asn Leu Gln Tyr Ile
325 330 335
Val Met His Ser Ile Ala Met Thr Ser Glu Thr Ala Ser Ser Thr Ile
340 345 350
Asp Gly Leu Lys Ala Thr Lys Asn Phe Leu His Cys Leu Gly Arg Tyr
355 360 365
Gly Asn Thr Pro Phe Leu Phe Pro Leu Tyr Gly Gln Gly Glu Leu Pro
370 375 380
Gln Cys Phe Cys Arg Met Cys Ala Val Phe Gly Gly Ile Tyr Cys Leu
385 390 395 400
Arg His Ser Val Gln Cys Leu Val Val Asp Lys Glu Ser Arg Lys Cys
405 410 415
Lys Ala Ile Ile Asp Gln Phe Gly Gln Arg Ile Ile Ser Glu His Phe
420 425 430
Leu Val Glu Asp Ser Tyr Phe Pro Glu Asn Met Cys Ser Arg Val Gln
435 440 445
Tyr Arg Gln Ile Ser Arg Ala Val Leu Ile Thr Asp Arg Ser Val Leu
450 455 460
Lys Thr Asp Ser Asp Gln Gln Ile Ser Ile Leu Thr Val Pro Ala Glu
465 470 475 480
Glu Pro Gly Thr Phe Ala Val Arg Val Ile Glu Leu Cys Ser Ser Thr
485 490 495
Met Thr Cys Met Lys Gly Thr Tyr Leu Val His Leu Thr Cys Thr Ser
500 505 510
Ser Lys Thr Ala Arg Glu Asp Leu Glu Ser Val Val Gln Lys Leu Phe
515 520 525
Val Pro Tyr Thr Glu Met Glu Ile Glu Asn Glu Gln Val Glu Lys Pro
530 535 540
Arg Ile Leu Trp Ala Leu Tyr Phe Asn Met Arg Asp Ser Ser Asp Ile
545 550 555 560
Ser Arg Ser Cys Tyr Asn Asp Leu Pro Ser Asn Val Tyr Val Cys Ser
565 570 575
Gly Pro Asp Cys Gly Leu Gly Asn Asp Asn Ala Val Lys Gln Ala Glu
580 585 590
Thr Leu Phe Gln Glu Ile Cys Pro Asn Glu Asp Phe Cys Pro Pro Pro
595 600 605
Pro Asn Pro Glu Asp Ile Ile Leu Asp Gly Asp Ser Leu Gln Pro Glu
610 615 620
Ala Ser Glu Ser Ser Ala Ile Pro Glu Ala Asn Ser Glu Thr Phe Lys
625 630 635 640
Glu Ser Thr Asn Leu Gly Asn Leu Glu Glu Ser Ser Glu
645 650
<210> 17
<211> 212
<212> PRT
<213> Homo sapiens
<400> 17
Met Ala Ser Leu Phe Ser Gly Arg Ile Leu Ile Arg Asn Asn Ser Asp
1 5 10 15
Gln Asp Glu Leu Asp Thr Glu Ala Glu Val Ser Arg Arg Leu Glu Asn
20 25 30
Arg Leu Val Leu Leu Phe Phe Gly Ala Gly Ala Cys Pro Gln Cys Gln
35 40 45
Ala Phe Val Pro Ile Leu Lys Asp Phe Phe Val Arg Leu Thr Asp Glu
50 55 60
Phe Tyr Val Leu Arg Ala Ala Gln Leu Ala Leu Val Tyr Val Ser Gln
65 70 75 80
Asp Ser Thr Glu Glu Gln Gln Asp Leu Phe Leu Lys Asp Met Pro Lys
85 90 95
Lys Trp Leu Phe Leu Pro Phe Glu Asp Asp Leu Arg Arg Asp Leu Gly
100 105 110
Arg Gln Phe Ser Val Glu Arg Leu Pro Ala Val Val Val Leu Lys Pro
115 120 125
Asp Gly Asp Val Leu Thr Arg Asp Gly Ala Asp Glu Ile Gln Arg Leu
130 135 140
Gly Thr Ala Cys Phe Ala Asn Trp Gln Glu Ala Ala Glu Val Leu Asp
145 150 155 160
Arg Asn Phe Gln Leu Pro Glu Asp Leu Glu Asp Gln Glu Pro Arg Ser
165 170 175
Leu Thr Glu Cys Leu Arg Arg His Lys Tyr Arg Val Glu Lys Ala Ala
180 185 190
Arg Gly Gly Arg Asp Pro Gly Gly Gly Gly Gly Glu Glu Gly Gly Ala
195 200 205
Gly Gly Leu Phe
210
<210> 18
<211> 156
<212> PRT
<213> Homo sapiens
<400> 18
Met Val Asp Ile Leu Gly Glu Arg His Leu Val Thr Cys Lys Gly Ala
1 5 10 15
Thr Val Glu Ala Glu Ala Ala Leu Gln Asn Lys Val Val Ala Leu Tyr
20 25 30
Phe Ala Ala Ala Arg Cys Ala Pro Ser Arg Asp Phe Thr Pro Leu Leu
35 40 45
Cys Asp Phe Tyr Thr Ala Leu Val Ala Glu Ala Arg Arg Pro Ala Pro
50 55 60
Phe Glu Val Val Phe Val Ser Ala Asp Gly Ser Ser Gln Glu Met Leu
65 70 75 80
Asp Phe Met Arg Glu Leu His Gly Ala Trp Leu Ala Leu Pro Phe His
85 90 95
Asp Pro Tyr Arg His Glu Leu Arg Lys Arg Tyr Asn Val Thr Ala Ile
100 105 110
Pro Lys Leu Val Ile Val Lys Gln Asn Gly Glu Val Ile Thr Asn Lys
115 120 125
Gly Arg Lys Gln Ile Arg Glu Arg Gly Leu Ala Cys Phe Gln Asp Trp
130 135 140
Val Glu Ala Ala Asp Ile Phe Gln Asn Phe Ser Val
145 150 155
<210> 19
<211> 135
<212> PRT
<213> Homo sapiens
<400> 19
Met Val Asp Ile Leu Gly Glu Arg His Leu Val Thr Cys Lys Gly Ala
1 5 10 15
Thr Val Glu Ala Glu Ala Ala Leu Gln Asn Lys Val Val Ala Leu Tyr
20 25 30
Phe Ala Ala Ala Arg Cys Ala Pro Ser Arg Asp Phe Thr Pro Leu Leu
35 40 45
Cys Asp Phe Tyr Thr Ala Leu Val Ala Glu Ala Arg Arg Pro Ala Pro
50 55 60
Phe Glu Val Val Phe Val Ser Ala Asp Gly Ser Ser Gln Glu Met Leu
65 70 75 80
Asp Phe Met Arg Glu Leu His Gly Ala Trp Leu Ala Leu Pro Phe His
85 90 95
Asp Pro Tyr Arg Gln Arg Ser Leu Ala Leu Leu Pro Arg Leu Glu Cys
100 105 110
Ser Gly Val Ile Leu Ala His Cys Asn Leu Cys Leu Leu Gly Ser Ser
115 120 125
Asp Ser Leu Ala Leu Ala Ser
130 135
<210> 20
<211> 860
<212> PRT
<213> Homo sapiens
<400> 20
Met Gly Glu Val Thr Ala Glu Glu Val Glu Lys Phe Leu Asp Ser Asn
1 5 10 15
Ile Gly Phe Ala Lys Gln Tyr Tyr Asn Leu His Tyr Arg Ala Lys Leu
20 25 30
Ile Ser Asp Leu Leu Gly Ala Lys Glu Ala Ala Val Asp Phe Ser Asn
35 40 45
Tyr His Ser Pro Ser Ser Met Glu Glu Ser Glu Ile Ile Phe Asp Leu
50 55 60
Leu Arg Asp Phe Gln Glu Asn Leu Gln Thr Glu Lys Cys Ile Phe Asn
65 70 75 80
Val Met Lys Lys Leu Cys Phe Leu Leu Gln Ala Asp Arg Met Ser Leu
85 90 95
Phe Met Tyr Arg Thr Arg Asn Gly Ile Ala Glu Leu Ala Thr Arg Leu
100 105 110
Phe Asn Val His Lys Asp Ala Val Leu Glu Asp Cys Leu Val Met Pro
115 120 125
Asp Gln Glu Ile Val Phe Pro Leu Asp Met Gly Ile Val Gly His Val
130 135 140
Ala His Ser Lys Lys Ile Ala Asn Val Pro Asn Thr Glu Glu Asp Glu
145 150 155 160
His Phe Cys Asp Phe Val Asp Ile Leu Thr Glu Tyr Lys Thr Lys Asn
165 170 175
Ile Leu Ala Ser Pro Ile Met Asn Gly Lys Asp Val Val Ala Ile Ile
180 185 190
Met Ala Val Asn Lys Val Asp Gly Ser His Phe Thr Lys Arg Asp Glu
195 200 205
Glu Ile Leu Leu Lys Tyr Leu Asn Phe Ala Asn Leu Ile Met Lys Val
210 215 220
Tyr His Leu Ser Tyr Leu His Asn Cys Glu Thr Arg Arg Gly Gln Ile
225 230 235 240
Leu Leu Trp Ser Gly Ser Lys Val Phe Glu Glu Leu Thr Asp Ile Glu
245 250 255
Arg Gln Phe His Lys Ala Leu Tyr Thr Val Arg Ala Phe Leu Asn Cys
260 265 270
Asp Arg Tyr Ser Val Gly Leu Leu Asp Met Thr Lys Gln Lys Glu Phe
275 280 285
Phe Asp Val Trp Pro Val Leu Met Gly Glu Val Pro Pro Tyr Ser Gly
290 295 300
Pro Arg Thr Pro Asp Gly Arg Glu Ile Asn Phe Tyr Lys Val Ile Asp
305 310 315 320
Tyr Ile Leu His Gly Lys Glu Asp Ile Lys Val Ile Pro Asn Pro Pro
325 330 335
Pro Asp His Trp Ala Leu Val Ser Gly Leu Pro Ala Tyr Val Ala Gln
340 345 350
Asn Gly Leu Ile Cys Asn Ile Met Asn Ala Pro Ala Glu Asp Phe Phe
355 360 365
Ala Phe Gln Lys Glu Pro Leu Asp Glu Ser Gly Trp Met Ile Lys Asn
370 375 380
Val Leu Ser Met Pro Ile Val Asn Lys Lys Glu Glu Ile Val Gly Val
385 390 395 400
Ala Thr Phe Tyr Asn Arg Lys Asp Gly Lys Pro Phe Asp Glu Met Asp
405 410 415
Glu Thr Leu Met Glu Ser Leu Thr Gln Phe Leu Gly Trp Ser Val Leu
420 425 430
Asn Pro Asp Thr Tyr Glu Ser Met Asn Lys Leu Glu Asn Arg Lys Asp
435 440 445
Ile Phe Gln Asp Ile Val Lys Tyr His Val Lys Cys Asp Asn Glu Glu
450 455 460
Ile Gln Lys Ile Leu Lys Thr Arg Glu Val Tyr Gly Lys Glu Pro Trp
465 470 475 480
Glu Cys Glu Glu Glu Glu Leu Ala Glu Ile Leu Gln Ala Glu Leu Pro
485 490 495
Asp Ala Asp Lys Tyr Glu Ile Asn Lys Phe His Phe Ser Asp Leu Pro
500 505 510
Leu Thr Glu Leu Glu Leu Val Lys Cys Gly Ile Gln Met Tyr Tyr Glu
515 520 525
Leu Lys Val Val Asp Lys Phe His Ile Pro Gln Glu Ala Leu Val Arg
530 535 540
Phe Met Tyr Ser Leu Ser Lys Gly Tyr Arg Lys Ile Thr Tyr His Asn
545 550 555 560
Trp Arg His Gly Phe Asn Val Gly Gln Thr Met Phe Ser Leu Leu Val
565 570 575
Thr Gly Lys Leu Lys Arg Tyr Phe Thr Asp Leu Glu Ala Leu Ala Met
580 585 590
Val Thr Ala Ala Phe Cys His Asp Ile Asp His Arg Gly Thr Asn Asn
595 600 605
Leu Tyr Gln Met Lys Ser Gln Asn Pro Leu Ala Lys Leu His Gly Ser
610 615 620
Ser Ile Leu Glu Arg His His Leu Glu Phe Gly Lys Thr Leu Leu Arg
625 630 635 640
Asp Glu Ser Leu Asn Ile Phe Gln Asn Leu Asn Arg Arg Gln His Glu
645 650 655
His Ala Ile His Met Met Asp Ile Ala Ile Ile Ala Thr Asp Leu Ala
660 665 670
Leu Tyr Phe Lys Lys Arg Thr Met Phe Gln Lys Ile Val Asp Gln Ser
675 680 685
Lys Thr Tyr Glu Ser Glu Gln Glu Trp Thr Gln Tyr Met Met Leu Glu
690 695 700
Gln Thr Arg Lys Glu Ile Val Met Ala Met Met Met Thr Ala Cys Asp
705 710 715 720
Leu Ser Ala Ile Thr Lys Pro Trp Glu Val Gln Ser Gln Val Ala Leu
725 730 735
Leu Val Ala Ala Glu Phe Trp Glu Gln Gly Asp Leu Glu Arg Thr Val
740 745 750
Leu Gln Gln Asn Pro Ile Pro Met Met Asp Arg Asn Lys Ala Asp Glu
755 760 765
Leu Pro Lys Leu Gln Val Gly Phe Ile Asp Phe Val Cys Thr Phe Val
770 775 780
Tyr Lys Glu Phe Ser Arg Phe His Glu Glu Ile Thr Pro Met Leu Asp
785 790 795 800
Gly Ile Thr Asn Asn Arg Lys Glu Trp Lys Ala Leu Ala Asp Glu Tyr
805 810 815
Asp Ala Lys Met Lys Val Gln Glu Glu Lys Lys Gln Lys Gln Gln Ser
820 825 830
Ala Lys Ser Ala Ala Ala Gly Asn Gln Pro Gly Gly Asn Pro Ser Pro
835 840 845
Gly Gly Ala Thr Thr Ser Lys Ser Cys Cys Ile Gln
850 855 860
<210> 21
<211> 854
<212> PRT
<213> Homo sapiens
<400> 21
Met Ser Leu Ser Glu Glu Gln Ala Arg Ser Phe Leu Asp Gln Asn Pro
1 5 10 15
Asp Phe Ala Arg Gln Tyr Phe Gly Lys Lys Leu Ser Pro Glu Asn Val
20 25 30
Ala Ala Ala Cys Glu Asp Gly Cys Pro Pro Asp Cys Asp Ser Leu Arg
35 40 45
Asp Leu Cys Gln Val Glu Glu Ser Thr Ala Leu Leu Glu Leu Val Gln
50 55 60
Asp Met Gln Glu Ser Ile Asn Met Glu Arg Val Val Phe Lys Val Leu
65 70 75 80
Arg Arg Leu Cys Thr Leu Leu Gln Ala Asp Arg Cys Ser Leu Phe Met
85 90 95
Tyr Arg Gln Arg Asn Gly Val Ala Glu Leu Ala Thr Arg Leu Phe Ser
100 105 110
Val Gln Pro Asp Ser Val Leu Glu Asp Cys Leu Val Pro Pro Asp Ser
115 120 125
Glu Ile Val Phe Pro Leu Asp Ile Gly Val Val Gly His Val Ala Gln
130 135 140
Thr Lys Lys Met Val Asn Val Glu Asp Val Ala Glu Cys Pro His Phe
145 150 155 160
Ser Ser Phe Ala Asp Glu Leu Thr Asp Tyr Lys Thr Lys Asn Met Leu
165 170 175
Ala Thr Pro Ile Met Asn Gly Lys Asp Val Val Ala Val Ile Met Ala
180 185 190
Val Asn Lys Leu Asn Gly Pro Phe Phe Thr Ser Glu Asp Glu Asp Val
195 200 205
Phe Leu Lys Tyr Leu Asn Phe Ala Thr Leu Tyr Leu Lys Ile Tyr His
210 215 220
Leu Ser Tyr Leu His Asn Cys Glu Thr Arg Arg Gly Gln Val Leu Leu
225 230 235 240
Trp Ser Ala Asn Lys Val Phe Glu Glu Leu Thr Asp Ile Glu Arg Gln
245 250 255
Phe His Lys Ala Phe Tyr Thr Val Arg Ala Tyr Leu Asn Cys Glu Arg
260 265 270
Tyr Ser Val Gly Leu Leu Asp Met Thr Lys Glu Lys Glu Phe Phe Asp
275 280 285
Val Trp Ser Val Leu Met Gly Glu Ser Gln Pro Tyr Ser Gly Pro Arg
290 295 300
Thr Pro Asp Gly Arg Glu Ile Val Phe Tyr Lys Val Ile Asp Tyr Ile
305 310 315 320
Leu His Gly Lys Glu Glu Ile Lys Val Ile Pro Thr Pro Ser Ala Asp
325 330 335
His Trp Ala Leu Ala Ser Gly Leu Pro Ser Tyr Val Ala Glu Ser Gly
340 345 350
Phe Ile Cys Asn Ile Met Asn Ala Ser Ala Asp Glu Met Phe Lys Phe
355 360 365
Gln Glu Gly Ala Leu Asp Asp Ser Gly Trp Leu Ile Lys Asn Val Leu
370 375 380
Ser Met Pro Ile Val Asn Lys Lys Glu Glu Ile Val Gly Val Ala Thr
385 390 395 400
Phe Tyr Asn Arg Lys Asp Gly Lys Pro Phe Asp Glu Gln Asp Glu Val
405 410 415
Leu Met Glu Ser Leu Thr Gln Phe Leu Gly Trp Ser Val Met Asn Thr
420 425 430
Asp Thr Tyr Asp Lys Met Asn Lys Leu Glu Asn Arg Lys Asp Ile Ala
435 440 445
Gln Asp Met Val Leu Tyr His Val Lys Cys Asp Arg Asp Glu Ile Gln
450 455 460
Leu Ile Leu Pro Thr Arg Ala Arg Leu Gly Lys Glu Pro Ala Asp Cys
465 470 475 480
Asp Glu Asp Glu Leu Gly Glu Ile Leu Lys Glu Glu Leu Pro Gly Pro
485 490 495
Thr Thr Phe Asp Ile Tyr Glu Phe His Phe Ser Asp Leu Glu Cys Thr
500 505 510
Glu Leu Asp Leu Val Lys Cys Gly Ile Gln Met Tyr Tyr Glu Leu Gly
515 520 525
Val Val Arg Lys Phe Gln Ile Pro Gln Glu Val Leu Val Arg Phe Leu
530 535 540
Phe Ser Ile Ser Lys Gly Tyr Arg Arg Ile Thr Tyr His Asn Trp Arg
545 550 555 560
His Gly Phe Asn Val Ala Gln Thr Met Phe Thr Leu Leu Met Thr Gly
565 570 575
Lys Leu Lys Ser Tyr Tyr Thr Asp Leu Glu Ala Phe Ala Met Val Thr
580 585 590
Ala Gly Leu Cys His Asp Ile Asp His Arg Gly Thr Asn Asn Leu Tyr
595 600 605
Gln Met Lys Ser Gln Asn Pro Leu Ala Lys Leu His Gly Ser Ser Ile
610 615 620
Leu Glu Arg His His Leu Glu Phe Gly Lys Phe Leu Leu Ser Glu Glu
625 630 635 640
Thr Leu Asn Ile Tyr Gln Asn Leu Asn Arg Arg Gln His Glu His Val
645 650 655
Ile His Leu Met Asp Ile Ala Ile Ile Ala Thr Asp Leu Ala Leu Tyr
660 665 670
Phe Lys Lys Arg Ala Met Phe Gln Lys Ile Val Asp Glu Ser Lys Asn
675 680 685
Tyr Gln Asp Lys Lys Ser Trp Val Glu Tyr Leu Ser Leu Glu Thr Thr
690 695 700
Arg Lys Glu Ile Val Met Ala Met Met Met Thr Ala Cys Asp Leu Ser
705 710 715 720
Ala Ile Thr Lys Pro Trp Glu Val Gln Ser Lys Val Ala Leu Leu Val
725 730 735
Ala Ala Glu Phe Trp Glu Gln Gly Asp Leu Glu Arg Thr Val Leu Asp
740 745 750
Gln Gln Pro Ile Pro Met Met Asp Arg Asn Lys Ala Ala Glu Leu Pro
755 760 765
Lys Leu Gln Val Gly Phe Ile Asp Phe Val Cys Thr Phe Val Tyr Lys
770 775 780
Glu Phe Ser Arg Phe His Glu Glu Ile Leu Pro Met Phe Asp Arg Leu
785 790 795 800
Gln Asn Asn Arg Lys Glu Trp Lys Ala Leu Ala Asp Glu Tyr Glu Ala
805 810 815
Lys Val Lys Ala Leu Glu Glu Lys Glu Glu Glu Glu Arg Val Ala Ala
820 825 830
Lys Lys Val Gly Thr Glu Ile Cys Asn Gly Gly Pro Ala Pro Lys Ser
835 840 845
Ser Thr Cys Cys Ile Leu
850
<210> 22
<211> 853
<212> PRT
<213> Homo sapiens
<400> 22
Met Ser Leu Ser Glu Glu Gln Ala Arg Ser Phe Leu Asp Gln Asn Pro
1 5 10 15
Asp Phe Ala Arg Gln Tyr Phe Gly Lys Lys Leu Ser Pro Glu Asn Val
20 25 30
Ala Ala Ala Cys Glu Asp Gly Cys Pro Pro Asp Cys Asp Ser Leu Arg
35 40 45
Asp Leu Cys Gln Val Glu Glu Ser Thr Ala Leu Leu Glu Leu Val Gln
50 55 60
Asp Met Gln Glu Ser Ile Asn Met Glu Arg Val Val Phe Lys Val Leu
65 70 75 80
Arg Arg Leu Cys Thr Leu Leu Gln Ala Asp Arg Cys Ser Leu Phe Met
85 90 95
Tyr Arg Gln Arg Asn Gly Val Ala Glu Leu Ala Thr Arg Leu Phe Ser
100 105 110
Val Gln Pro Asp Ser Val Leu Glu Asp Cys Leu Val Pro Pro Asp Ser
115 120 125
Glu Ile Val Phe Pro Leu Asp Ile Gly Val Val Gly His Val Ala Gln
130 135 140
Thr Lys Lys Met Val Asn Val Glu Asp Val Ala Glu Cys Pro His Phe
145 150 155 160
Ser Ser Phe Ala Asp Glu Leu Thr Asp Tyr Lys Thr Lys Asn Met Leu
165 170 175
Ala Thr Pro Ile Met Asn Gly Lys Asp Val Val Ala Val Ile Met Ala
180 185 190
Val Asn Lys Leu Asn Gly Pro Phe Phe Thr Ser Glu Asp Glu Asp Val
195 200 205
Phe Leu Lys Tyr Leu Asn Phe Ala Thr Leu Tyr Leu Lys Ile Tyr His
210 215 220
Leu Ser Tyr Leu His Asn Cys Glu Thr Arg Arg Gly Gln Val Leu Leu
225 230 235 240
Trp Ser Ala Asn Lys Val Phe Glu Glu Leu Thr Asp Ile Glu Arg Gln
245 250 255
Phe His Lys Ala Phe Tyr Thr Val Arg Ala Tyr Leu Asn Cys Glu Arg
260 265 270
Tyr Ser Val Gly Leu Leu Asp Met Thr Lys Glu Lys Glu Phe Phe Asp
275 280 285
Val Trp Ser Val Leu Met Gly Glu Ser Gln Pro Tyr Ser Gly Pro Arg
290 295 300
Thr Pro Asp Gly Arg Glu Ile Val Phe Tyr Lys Val Ile Asp Tyr Ile
305 310 315 320
Leu His Gly Lys Glu Glu Ile Lys Val Ile Pro Thr Pro Ser Ala Asp
325 330 335
His Trp Ala Leu Ala Ser Gly Leu Pro Ser Tyr Val Ala Glu Ser Gly
340 345 350
Phe Ile Cys Asn Ile Met Asn Ala Ser Ala Asp Glu Met Phe Lys Phe
355 360 365
Gln Glu Gly Ala Leu Asp Asp Ser Gly Trp Leu Ile Lys Asn Val Leu
370 375 380
Ser Met Pro Ile Val Asn Lys Lys Glu Glu Ile Val Gly Val Ala Thr
385 390 395 400
Phe Tyr Asn Arg Lys Asp Gly Lys Pro Phe Asp Glu Gln Asp Glu Val
405 410 415
Leu Met Glu Ser Leu Thr Gln Phe Leu Gly Trp Ser Val Met Asn Thr
420 425 430
Asp Thr Tyr Asp Lys Met Asn Lys Leu Glu Asn Arg Lys Asp Ile Ala
435 440 445
Gln Asp Met Val Leu Tyr His Val Lys Cys Asp Arg Asp Glu Ile Gln
450 455 460
Leu Ile Leu Pro Thr Arg Ala Arg Leu Gly Lys Glu Pro Ala Asp Cys
465 470 475 480
Asp Glu Asp Glu Leu Gly Glu Ile Leu Lys Glu Glu Leu Pro Gly Pro
485 490 495
Thr Thr Phe Asp Ile Tyr Glu Phe His Phe Ser Asp Leu Glu Cys Thr
500 505 510
Glu Leu Asp Leu Val Lys Cys Gly Ile Gln Met Tyr Tyr Glu Leu Gly
515 520 525
Val Val Arg Lys Phe Gln Ile Pro Gln Glu Val Leu Val Arg Phe Leu
530 535 540
Phe Ser Ile Ser Lys Gly Tyr Arg Arg Ile Thr Tyr His Asn Trp Arg
545 550 555 560
His Gly Phe Asn Val Ala Gln Thr Met Phe Thr Leu Leu Met Thr Gly
565 570 575
Lys Leu Lys Ser Tyr Tyr Thr Asp Leu Glu Ala Phe Ala Met Val Thr
580 585 590
Ala Gly Leu Cys His Asp Ile Asp His Arg Gly Thr Asn Asn Leu Tyr
595 600 605
Gln Met Lys Ser Gln Asn Pro Leu Ala Lys Leu His Gly Ser Ser Ile
610 615 620
Leu Glu Arg His His Leu Glu Phe Gly Lys Phe Leu Leu Ser Glu Glu
625 630 635 640
Thr Leu Asn Ile Tyr Gln Asn Leu Asn Arg Arg Gln His Glu His Val
645 650 655
Ile His Leu Met Asp Ile Ala Ile Ile Ala Thr Asp Leu Ala Leu Tyr
660 665 670
Phe Lys Lys Arg Ala Met Phe Gln Lys Ile Val Asp Glu Ser Lys Asn
675 680 685
Tyr Gln Asp Lys Lys Ser Trp Val Glu Tyr Leu Ser Leu Glu Thr Thr
690 695 700
Arg Lys Glu Ile Val Met Ala Met Met Met Thr Ala Cys Asp Leu Ser
705 710 715 720
Ala Ile Thr Lys Pro Trp Glu Val Gln Ser Lys Val Ala Leu Leu Val
725 730 735
Ala Ala Glu Phe Trp Glu Gln Gly Asp Leu Glu Arg Thr Val Leu Asp
740 745 750
Gln Gln Pro Ile Pro Met Met Asp Arg Asn Lys Ala Ala Glu Leu Pro
755 760 765
Lys Leu Gln Val Gly Phe Ile Asp Phe Val Cys Thr Phe Val Tyr Lys
770 775 780
Glu Phe Ser Arg Phe His Glu Glu Ile Leu Pro Met Phe Asp Arg Leu
785 790 795 800
Gln Asn Asn Arg Lys Glu Trp Lys Ala Leu Ala Asp Glu Tyr Glu Ala
805 810 815
Lys Val Lys Ala Leu Glu Glu Lys Glu Glu Glu Glu Arg Val Ala Ala
820 825 830
Lys Lys Gly Thr Glu Ile Cys Asn Gly Gly Pro Ala Pro Lys Ser Ser
835 840 845
Thr Cys Cys Ile Leu
850
<210> 23
<211> 575
<212> PRT
<213> Homo sapiens
<400> 23
Met Thr Lys Glu Lys Glu Phe Phe Asp Val Trp Ser Val Leu Met Gly
1 5 10 15
Glu Ser Gln Pro Tyr Ser Gly Pro Arg Thr Pro Asp Gly Arg Glu Ile
20 25 30
Val Phe Tyr Lys Val Ile Asp Tyr Ile Leu His Gly Lys Glu Glu Ile
35 40 45
Lys Val Ile Pro Thr Pro Ser Ala Asp His Trp Ala Leu Ala Ser Gly
50 55 60
Leu Pro Ser Tyr Val Ala Glu Ser Gly Phe Ile Cys Asn Ile Met Asn
65 70 75 80
Ala Ser Ala Asp Glu Met Phe Lys Phe Gln Glu Gly Ala Leu Asp Asp
85 90 95
Ser Gly Trp Leu Ile Lys Asn Val Leu Ser Met Pro Ile Val Asn Lys
100 105 110
Lys Glu Glu Ile Val Gly Val Ala Thr Phe Tyr Asn Arg Lys Asp Gly
115 120 125
Lys Pro Phe Asp Glu Gln Asp Glu Val Leu Met Glu Ser Leu Thr Gln
130 135 140
Phe Leu Gly Trp Ser Val Met Asn Thr Asp Thr Tyr Asp Lys Met Asn
145 150 155 160
Lys Leu Glu Asn Arg Lys Asp Ile Ala Gln Asp Met Val Leu Tyr His
165 170 175
Val Lys Cys Asp Arg Asp Glu Ile Gln Leu Ile Leu Pro Thr Arg Ala
180 185 190
Arg Leu Gly Lys Glu Pro Ala Asp Cys Asp Glu Asp Glu Leu Gly Glu
195 200 205
Ile Leu Lys Glu Glu Leu Pro Gly Pro Thr Thr Phe Asp Ile Tyr Glu
210 215 220
Phe His Phe Ser Asp Leu Glu Cys Thr Glu Leu Asp Leu Val Lys Cys
225 230 235 240
Gly Ile Gln Met Tyr Tyr Glu Leu Gly Val Val Arg Lys Phe Gln Ile
245 250 255
Pro Gln Glu Val Leu Val Arg Phe Leu Phe Ser Ile Ser Lys Gly Tyr
260 265 270
Arg Arg Ile Thr Tyr His Asn Trp Arg His Gly Phe Asn Val Ala Gln
275 280 285
Thr Met Phe Thr Leu Leu Met Thr Gly Lys Leu Lys Ser Tyr Tyr Thr
290 295 300
Asp Leu Glu Ala Phe Ala Met Val Thr Ala Gly Leu Cys His Asp Ile
305 310 315 320
Asp His Arg Gly Thr Asn Asn Leu Tyr Gln Met Lys Ser Gln Asn Pro
325 330 335
Leu Ala Lys Leu His Gly Ser Ser Ile Leu Glu Arg His His Leu Glu
340 345 350
Phe Gly Lys Phe Leu Leu Ser Glu Glu Thr Leu Asn Ile Tyr Gln Asn
355 360 365
Leu Asn Arg Arg Gln His Glu His Val Ile His Leu Met Asp Ile Ala
370 375 380
Ile Ile Ala Thr Asp Leu Ala Leu Tyr Phe Lys Lys Arg Ala Met Phe
385 390 395 400
Gln Lys Ile Val Asp Glu Ser Lys Asn Tyr Gln Asp Lys Lys Ser Trp
405 410 415
Val Glu Tyr Leu Ser Leu Glu Thr Thr Arg Lys Glu Ile Val Met Ala
420 425 430
Met Met Met Thr Ala Cys Asp Leu Ser Ala Ile Thr Lys Pro Trp Glu
435 440 445
Val Gln Ser Lys Val Ala Leu Leu Val Ala Ala Glu Phe Trp Glu Gln
450 455 460
Gly Asp Leu Glu Arg Thr Val Leu Asp Gln Gln Pro Ile Pro Met Met
465 470 475 480
Asp Arg Asn Lys Ala Ala Glu Leu Pro Lys Leu Gln Val Gly Phe Ile
485 490 495
Asp Phe Val Cys Thr Phe Val Tyr Lys Glu Phe Ser Arg Phe His Glu
500 505 510
Glu Ile Leu Pro Met Phe Asp Arg Leu Gln Asn Asn Arg Lys Glu Trp
515 520 525
Lys Ala Leu Ala Asp Glu Tyr Glu Ala Lys Val Lys Ala Leu Glu Glu
530 535 540
Lys Glu Glu Glu Glu Arg Val Ala Ala Lys Lys Val Gly Thr Glu Ile
545 550 555 560
Cys Asn Gly Gly Pro Ala Pro Lys Ser Ser Thr Cys Cys Ile Leu
565 570 575
<210> 24
<211> 694
<212> PRT
<213> Homo sapiens
<400> 24
Met Ala Lys Ile Asn Thr Gln Tyr Ser His Pro Ser Arg Thr His Leu
1 5 10 15
Lys Val Lys Thr Ser Asp Arg Asp Leu Asn Arg Ala Glu Asn Gly Leu
20 25 30
Ser Arg Ala His Ser Ser Ser Glu Glu Thr Ser Ser Val Leu Gln Pro
35 40 45
Gly Ile Ala Met Glu Thr Arg Gly Leu Ala Asp Ser Gly Gln Gly Ser
50 55 60
Phe Thr Gly Gln Gly Ile Ala Arg Leu Ser Arg Leu Ile Phe Leu Leu
65 70 75 80
Arg Arg Trp Ala Ala Arg His Val His His Gln Asp Gln Gly Pro Asp
85 90 95
Ser Phe Pro Asp Arg Phe Arg Gly Ala Glu Leu Lys Glu Val Ser Ser
100 105 110
Gln Glu Ser Asn Ala Gln Ala Asn Val Gly Ser Gln Glu Pro Ala Asp
115 120 125
Arg Gly Arg Ser Ala Trp Pro Leu Ala Lys Cys Asn Thr Asn Thr Ser
130 135 140
Asn Asn Thr Glu Glu Glu Lys Lys Thr Lys Lys Lys Asp Ala Ile Val
145 150 155 160
Val Asp Pro Ser Ser Asn Leu Tyr Tyr Arg Trp Leu Thr Ala Ile Ala
165 170 175
Leu Pro Val Phe Tyr Asn Trp Tyr Leu Leu Ile Cys Arg Ala Cys Phe
180 185 190
Asp Glu Leu Gln Ser Glu Tyr Leu Met Leu Trp Leu Val Leu Asp Tyr
195 200 205
Ser Ala Asp Val Leu Tyr Val Leu Asp Val Leu Val Arg Ala Arg Thr
210 215 220
Gly Phe Leu Glu Gln Gly Leu Met Val Ser Asp Thr Asn Arg Leu Trp
225 230 235 240
Gln His Tyr Lys Thr Thr Thr Gln Phe Lys Leu Asp Val Leu Ser Leu
245 250 255
Val Pro Thr Asp Leu Ala Tyr Leu Lys Val Gly Thr Asn Tyr Pro Glu
260 265 270
Val Arg Phe Asn Arg Leu Leu Lys Phe Ser Arg Leu Phe Glu Phe Phe
275 280 285
Asp Arg Thr Glu Thr Arg Thr Asn Tyr Pro Asn Met Phe Arg Ile Gly
290 295 300
Asn Leu Val Leu Tyr Ile Leu Ile Ile Ile His Trp Asn Ala Cys Ile
305 310 315 320
Tyr Phe Ala Ile Ser Lys Phe Ile Gly Phe Gly Thr Asp Ser Trp Val
325 330 335
Tyr Pro Asn Ile Ser Ile Pro Glu His Gly Arg Leu Ser Arg Lys Tyr
340 345 350
Ile Tyr Ser Leu Tyr Trp Ser Thr Leu Thr Leu Thr Thr Ile Gly Glu
355 360 365
Thr Pro Pro Pro Val Lys Asp Glu Glu Tyr Leu Phe Val Val Val Asp
370 375 380
Phe Leu Val Gly Val Leu Ile Phe Ala Thr Ile Val Gly Asn Val Gly
385 390 395 400
Ser Met Ile Ser Asn Met Asn Ala Ser Arg Ala Glu Phe Gln Ala Lys
405 410 415
Ile Asp Ser Ile Lys Gln Tyr Met Gln Phe Arg Lys Val Thr Lys Asp
420 425 430
Leu Glu Thr Arg Val Ile Arg Trp Phe Asp Tyr Leu Trp Ala Asn Lys
435 440 445
Lys Thr Val Asp Glu Lys Glu Val Leu Lys Ser Leu Pro Asp Lys Leu
450 455 460
Lys Ala Glu Ile Ala Ile Asn Val His Leu Asp Thr Leu Lys Lys Val
465 470 475 480
Arg Ile Phe Gln Asp Cys Glu Ala Gly Leu Leu Val Glu Leu Val Leu
485 490 495
Lys Leu Arg Pro Thr Val Phe Ser Pro Gly Asp Tyr Ile Cys Lys Lys
500 505 510
Gly Asp Ile Gly Lys Glu Met Tyr Ile Ile Asn Glu Gly Lys Leu Ala
515 520 525
Val Val Ala Asp Asp Gly Val Thr Gln Phe Val Val Leu Ser Asp Gly
530 535 540
Ser Tyr Phe Gly Glu Ile Ser Ile Leu Asn Ile Lys Gly Ser Lys Ser
545 550 555 560
Gly Asn Arg Arg Thr Ala Asn Ile Arg Ser Ile Gly Tyr Ser Asp Leu
565 570 575
Phe Cys Leu Ser Lys Asp Asp Leu Met Glu Ala Leu Thr Glu Tyr Pro
580 585 590
Glu Ala Lys Lys Ala Leu Glu Glu Lys Gly Arg Gln Ile Leu Met Lys
595 600 605
Asp Asn Leu Ile Asp Glu Glu Leu Ala Arg Ala Gly Ala Asp Pro Lys
610 615 620
Asp Leu Glu Glu Lys Val Glu Gln Leu Gly Ser Ser Leu Asp Thr Leu
625 630 635 640
Gln Thr Arg Phe Ala Arg Leu Leu Ala Glu Tyr Asn Ala Thr Gln Met
645 650 655
Lys Met Lys Gln Arg Leu Ser Gln Leu Glu Ser Gln Val Lys Gly Gly
660 665 670
Gly Asp Lys Pro Leu Ala Asp Gly Glu Val Pro Gly Asp Ala Thr Lys
675 680 685
Thr Glu Asp Lys Gln Gln
690
<210> 25
<211> 676
<212> PRT
<213> Homo sapiens
<400> 25
Met Ala Lys Ile Asn Thr Gln Tyr Ser His Pro Ser Arg Thr His Leu
1 5 10 15
Lys Val Lys Thr Ser Asp Arg Asp Leu Asn Arg Ala Glu Asn Gly Leu
20 25 30
Ser Arg Ala His Ser Ser Ser Glu Glu Thr Ser Ser Val Leu Gln Pro
35 40 45
Gly Ile Ala Met Glu Thr Arg Gly Leu Ala Asp Ser Gly Gln Gly Ser
50 55 60
Phe Thr Gly Gln Gly Ile Ala Arg Leu Ser Arg Leu Ile Phe Leu Leu
65 70 75 80
Arg Arg Trp Ala Ala Arg His Val His His Gln Asp Gln Gly Pro Asp
85 90 95
Ser Phe Pro Asp Arg Phe Arg Gly Ala Glu Leu Lys Glu Val Ser Ser
100 105 110
Gln Glu Ser Asn Ala Gln Ala Asn Val Gly Ser Gln Glu Pro Ala Asp
115 120 125
Arg Gly Arg Arg Lys Lys Thr Lys Lys Lys Asp Ala Ile Val Val Asp
130 135 140
Pro Ser Ser Asn Leu Tyr Tyr Arg Trp Leu Thr Ala Ile Ala Leu Pro
145 150 155 160
Val Phe Tyr Asn Trp Tyr Leu Leu Ile Cys Arg Ala Cys Phe Asp Glu
165 170 175
Leu Gln Ser Glu Tyr Leu Met Leu Trp Leu Val Leu Asp Tyr Ser Ala
180 185 190
Asp Val Leu Tyr Val Leu Asp Val Leu Val Arg Ala Arg Thr Gly Phe
195 200 205
Leu Glu Gln Gly Leu Met Val Ser Asp Thr Asn Arg Leu Trp Gln His
210 215 220
Tyr Lys Thr Thr Thr Gln Phe Lys Leu Asp Val Leu Ser Leu Val Pro
225 230 235 240
Thr Asp Leu Ala Tyr Leu Lys Val Gly Thr Asn Tyr Pro Glu Val Arg
245 250 255
Phe Asn Arg Leu Leu Lys Phe Ser Arg Leu Phe Glu Phe Phe Asp Arg
260 265 270
Thr Glu Thr Arg Thr Asn Tyr Pro Asn Met Phe Arg Ile Gly Asn Leu
275 280 285
Val Leu Tyr Ile Leu Ile Ile Ile His Trp Asn Ala Cys Ile Tyr Phe
290 295 300
Ala Ile Ser Lys Phe Ile Gly Phe Gly Thr Asp Ser Trp Val Tyr Pro
305 310 315 320
Asn Ile Ser Ile Pro Glu His Gly Arg Leu Ser Arg Lys Tyr Ile Tyr
325 330 335
Ser Leu Tyr Trp Ser Thr Leu Thr Leu Thr Thr Ile Gly Glu Thr Pro
340 345 350
Pro Pro Val Lys Asp Glu Glu Tyr Leu Phe Val Val Val Asp Phe Leu
355 360 365
Val Gly Val Leu Ile Phe Ala Thr Ile Val Gly Asn Val Gly Ser Met
370 375 380
Ile Ser Asn Met Asn Ala Ser Arg Ala Glu Phe Gln Ala Lys Ile Asp
385 390 395 400
Ser Ile Lys Gln Tyr Met Gln Phe Arg Lys Val Thr Lys Asp Leu Glu
405 410 415
Thr Arg Val Ile Arg Trp Phe Asp Tyr Leu Trp Ala Asn Lys Lys Thr
420 425 430
Val Asp Glu Lys Glu Val Leu Lys Ser Leu Pro Asp Lys Leu Lys Ala
435 440 445
Glu Ile Ala Ile Asn Val His Leu Asp Thr Leu Lys Lys Val Arg Ile
450 455 460
Phe Gln Asp Cys Glu Ala Gly Leu Leu Val Glu Leu Val Leu Lys Leu
465 470 475 480
Arg Pro Thr Val Phe Ser Pro Gly Asp Tyr Ile Cys Lys Lys Gly Asp
485 490 495
Ile Gly Lys Glu Met Tyr Ile Ile Asn Glu Gly Lys Leu Ala Val Val
500 505 510
Ala Asp Asp Gly Val Thr Gln Phe Val Val Leu Ser Asp Gly Ser Tyr
515 520 525
Phe Gly Glu Ile Ser Ile Leu Asn Ile Lys Gly Ser Lys Ser Gly Asn
530 535 540
Arg Arg Thr Ala Asn Ile Arg Ser Ile Gly Tyr Ser Asp Leu Phe Cys
545 550 555 560
Leu Ser Lys Asp Asp Leu Met Glu Ala Leu Thr Glu Tyr Pro Glu Ala
565 570 575
Lys Lys Ala Leu Glu Glu Lys Gly Arg Gln Ile Leu Met Lys Asp Asn
580 585 590
Leu Ile Asp Glu Glu Leu Ala Arg Ala Gly Ala Asp Pro Lys Asp Leu
595 600 605
Glu Glu Lys Val Glu Gln Leu Gly Ser Ser Leu Asp Thr Leu Gln Thr
610 615 620
Arg Phe Ala Arg Leu Leu Ala Glu Tyr Asn Ala Thr Gln Met Lys Met
625 630 635 640
Lys Gln Arg Leu Ser Gln Leu Glu Ser Gln Val Lys Gly Gly Gly Asp
645 650 655
Lys Pro Leu Ala Asp Gly Glu Val Pro Gly Asp Ala Thr Lys Thr Glu
660 665 670
Asp Lys Gln Gln
675
<210> 26
<211> 809
<212> PRT
<213> Homo sapiens
<400> 26
Met Phe Lys Ser Leu Thr Lys Val Asn Lys Val Lys Pro Ile Gly Glu
1 5 10 15
Asn Asn Glu Asn Glu Gln Ser Ser Arg Arg Asn Glu Glu Gly Ser His
20 25 30
Pro Ser Asn Gln Ser Gln Gln Thr Thr Ala Gln Glu Glu Asn Lys Gly
35 40 45
Glu Glu Lys Ser Leu Lys Thr Lys Ser Thr Pro Val Thr Ser Glu Glu
50 55 60
Pro His Thr Asn Ile Gln Asp Lys Leu Ser Lys Lys Asn Ser Ser Gly
65 70 75 80
Asp Leu Thr Thr Asn Pro Asp Pro Gln Asn Ala Ala Glu Pro Thr Gly
85 90 95
Thr Val Pro Glu Gln Lys Glu Met Asp Pro Gly Lys Glu Gly Pro Asn
100 105 110
Ser Pro Gln Asn Lys Pro Pro Ala Ala Pro Val Ile Asn Glu Tyr Ala
115 120 125
Asp Ala Gln Leu His Asn Leu Val Lys Arg Met Arg Gln Arg Thr Ala
130 135 140
Leu Tyr Lys Lys Lys Leu Val Glu Gly Asp Leu Ser Ser Pro Glu Ala
145 150 155 160
Ser Pro Gln Thr Ala Lys Pro Thr Ala Val Pro Pro Val Lys Glu Ser
165 170 175
Asp Asp Lys Pro Thr Glu His Tyr Tyr Arg Leu Leu Trp Phe Lys Val
180 185 190
Lys Lys Met Pro Leu Thr Glu Tyr Leu Lys Arg Ile Lys Leu Pro Asn
195 200 205
Ser Ile Asp Ser Tyr Thr Asp Arg Leu Tyr Leu Leu Trp Leu Leu Leu
210 215 220
Val Thr Leu Ala Tyr Asn Trp Asn Cys Cys Phe Ile Pro Leu Arg Leu
225 230 235 240
Val Phe Pro Tyr Gln Thr Ala Asp Asn Ile His Tyr Trp Leu Ile Ala
245 250 255
Asp Ile Ile Cys Asp Ile Ile Tyr Leu Tyr Asp Met Leu Phe Ile Gln
260 265 270
Pro Arg Leu Gln Phe Val Arg Gly Gly Asp Ile Ile Val Asp Ser Asn
275 280 285
Glu Leu Arg Lys His Tyr Arg Thr Ser Thr Lys Phe Gln Leu Asp Val
290 295 300
Ala Ser Ile Ile Pro Phe Asp Ile Cys Tyr Leu Phe Phe Gly Phe Asn
305 310 315 320
Pro Met Phe Arg Ala Asn Arg Met Leu Lys Tyr Thr Ser Phe Phe Glu
325 330 335
Phe Asn His His Leu Glu Ser Ile Met Asp Lys Ala Tyr Ile Tyr Arg
340 345 350
Val Ile Arg Thr Thr Gly Tyr Leu Leu Phe Ile Leu His Ile Asn Ala
355 360 365
Cys Val Tyr Tyr Trp Ala Ser Asn Tyr Glu Gly Ile Gly Thr Thr Arg
370 375 380
Trp Val Tyr Asp Gly Glu Gly Asn Glu Tyr Leu Arg Cys Tyr Tyr Trp
385 390 395 400
Ala Val Arg Thr Leu Ile Thr Ile Gly Gly Leu Pro Glu Pro Gln Thr
405 410 415
Leu Phe Glu Ile Val Phe Gln Leu Leu Asn Phe Phe Ser Gly Val Phe
420 425 430
Val Phe Ser Ser Leu Ile Gly Gln Met Arg Asp Val Ile Gly Ala Ala
435 440 445
Thr Ala Asn Gln Asn Tyr Phe Arg Ala Cys Met Asp Asp Thr Ile Ala
450 455 460
Tyr Met Asn Asn Tyr Ser Ile Pro Lys Leu Val Gln Lys Arg Val Arg
465 470 475 480
Thr Trp Tyr Glu Tyr Thr Trp Asp Ser Gln Arg Met Leu Asp Glu Ser
485 490 495
Asp Leu Leu Lys Thr Leu Pro Thr Thr Val Gln Leu Ala Leu Ala Ile
500 505 510
Asp Val Asn Phe Ser Ile Ile Ser Lys Val Asp Leu Phe Lys Gly Cys
515 520 525
Asp Thr Gln Met Ile Tyr Asp Met Leu Leu Arg Leu Lys Ser Val Leu
530 535 540
Tyr Leu Pro Gly Asp Phe Val Cys Lys Lys Gly Glu Ile Gly Lys Glu
545 550 555 560
Met Tyr Ile Ile Lys His Gly Glu Val Gln Val Leu Gly Gly Pro Asp
565 570 575
Gly Thr Lys Val Leu Val Thr Leu Lys Ala Gly Ser Val Phe Gly Glu
580 585 590
Ile Ser Leu Leu Ala Ala Gly Gly Gly Asn Arg Arg Thr Ala Asn Val
595 600 605
Val Ala His Gly Phe Ala Asn Leu Leu Thr Leu Asp Lys Lys Thr Leu
610 615 620
Gln Glu Ile Leu Val His Tyr Pro Asp Ser Glu Arg Ile Leu Met Lys
625 630 635 640
Lys Ala Arg Val Leu Leu Lys Gln Lys Ala Lys Thr Ala Glu Ala Thr
645 650 655
Pro Pro Arg Lys Asp Leu Ala Leu Leu Phe Pro Pro Lys Glu Glu Thr
660 665 670
Pro Lys Leu Phe Lys Thr Leu Leu Gly Gly Thr Gly Lys Ala Ser Leu
675 680 685
Ala Arg Leu Leu Lys Leu Lys Arg Glu Gln Ala Ala Gln Lys Lys Glu
690 695 700
Asn Ser Glu Gly Gly Glu Glu Glu Gly Lys Glu Asn Glu Asp Lys Gln
705 710 715 720
Lys Glu Asn Glu Asp Lys Gln Lys Glu Asn Glu Asp Lys Gly Lys Glu
725 730 735
Asn Glu Asp Lys Asp Lys Gly Arg Glu Pro Glu Glu Lys Pro Leu Asp
740 745 750
Arg Pro Glu Cys Thr Ala Ser Pro Ile Ala Val Glu Glu Glu Pro His
755 760 765
Ser Val Arg Arg Thr Val Leu Pro Arg Gly Thr Ser Arg Gln Ser Leu
770 775 780
Ile Ile Ser Met Ala Pro Ser Ala Glu Gly Gly Glu Glu Val Leu Thr
785 790 795 800
Ile Glu Val Lys Glu Lys Ala Lys Gln
805
<210> 27
<211> 354
<212> PRT
<213> Homo sapiens
<400> 27
Met Gly Ser Gly Ala Ser Ala Glu Asp Lys Glu Leu Ala Lys Arg Ser
1 5 10 15
Lys Glu Leu Glu Lys Lys Leu Gln Glu Asp Ala Asp Lys Glu Ala Lys
20 25 30
Thr Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr
35 40 45
Ile Val Lys Gln Met Lys Ile Ile His Gln Asp Gly Tyr Ser Pro Glu
50 55 60
Glu Cys Leu Glu Phe Lys Ala Ile Ile Tyr Gly Asn Val Leu Gln Ser
65 70 75 80
Ile Leu Ala Ile Ile Arg Ala Met Thr Thr Leu Gly Ile Asp Tyr Ala
85 90 95
Glu Pro Ser Cys Ala Asp Asp Gly Arg Gln Leu Asn Asn Leu Ala Asp
100 105 110
Ser Ile Glu Glu Gly Thr Met Pro Pro Glu Leu Val Glu Val Ile Arg
115 120 125
Arg Leu Trp Lys Asp Gly Gly Val Gln Ala Cys Phe Glu Arg Ala Ala
130 135 140
Glu Tyr Gln Leu Asn Asp Ser Ala Ser Tyr Tyr Leu Asn Gln Leu Glu
145 150 155 160
Arg Ile Thr Asp Pro Glu Tyr Leu Pro Ser Glu Gln Asp Val Leu Arg
165 170 175
Ser Arg Val Lys Thr Thr Gly Ile Ile Glu Thr Lys Phe Ser Val Lys
180 185 190
Asp Leu Asn Phe Arg Met Phe Asp Val Gly Gly Gln Arg Ser Glu Arg
195 200 205
Lys Lys Trp Ile His Cys Phe Glu Gly Val Thr Cys Ile Ile Phe Cys
210 215 220
Ala Ala Leu Ser Ala Tyr Asp Met Val Leu Val Glu Asp Asp Glu Val
225 230 235 240
Asn Arg Met His Glu Ser Leu His Leu Phe Asn Ser Ile Cys Asn His
245 250 255
Lys Phe Phe Ala Ala Thr Ser Ile Val Leu Phe Leu Asn Lys Lys Asp
260 265 270
Leu Phe Glu Glu Lys Ile Lys Lys Val His Leu Ser Ile Cys Phe Pro
275 280 285
Glu Tyr Asp Gly Asn Asn Ser Tyr Asp Asp Ala Gly Asn Tyr Ile Lys
290 295 300
Ser Gln Phe Leu Asp Leu Asn Met Arg Lys Asp Val Lys Glu Ile Tyr
305 310 315 320
Ser His Met Thr Cys Ala Thr Asp Thr Gln Asn Val Lys Phe Val Phe
325 330 335
Asp Ala Val Thr Asp Ile Ile Ile Lys Glu Asn Leu Lys Asp Cys Gly
340 345 350
Leu Phe
<210> 28
<211> 815
<212> PRT
<213> Homo sapiens
<400> 28
Met Arg Glu Pro Glu Glu Leu Met Pro Asp Ser Gly Ala Val Phe Thr
1 5 10 15
Phe Gly Lys Ser Lys Phe Ala Glu Asn Asn Pro Gly Lys Phe Trp Phe
20 25 30
Lys Asn Asp Val Pro Val His Leu Ser Cys Gly Asp Glu His Ser Ala
35 40 45
Val Val Thr Gly Asn Asn Lys Leu Tyr Met Phe Gly Ser Asn Asn Trp
50 55 60
Gly Gln Leu Gly Leu Gly Ser Lys Ser Ala Ile Ser Lys Pro Thr Cys
65 70 75 80
Val Lys Ala Leu Lys Pro Glu Lys Val Lys Leu Ala Ala Cys Gly Arg
85 90 95
Asn His Thr Leu Val Ser Thr Glu Gly Gly Asn Val Tyr Ala Thr Gly
100 105 110
Gly Asn Asn Glu Gly Gln Leu Gly Leu Gly Asp Thr Glu Glu Arg Asn
115 120 125
Thr Phe His Val Ile Ser Phe Phe Thr Ser Glu His Lys Ile Lys Gln
130 135 140
Leu Ser Ala Gly Ser Asn Thr Ser Ala Ala Leu Thr Glu Asp Gly Arg
145 150 155 160
Leu Phe Met Trp Gly Asp Asn Ser Glu Gly Gln Ile Gly Leu Lys Asn
165 170 175
Val Ser Asn Val Cys Val Pro Gln Gln Val Thr Ile Gly Lys Pro Val
180 185 190
Ser Trp Ile Ser Cys Gly Tyr Tyr His Ser Ala Phe Val Thr Thr Asp
195 200 205
Gly Glu Leu Tyr Val Phe Gly Glu Pro Glu Asn Gly Lys Leu Gly Leu
210 215 220
Pro Asn Gln Leu Leu Gly Asn His Arg Thr Pro Gln Leu Val Ser Glu
225 230 235 240
Ile Pro Glu Lys Val Ile Gln Val Ala Cys Gly Gly Glu His Thr Val
245 250 255
Val Leu Thr Glu Asn Ala Val Tyr Thr Phe Gly Leu Gly Gln Phe Gly
260 265 270
Gln Leu Gly Leu Gly Thr Phe Leu Phe Glu Thr Ser Glu Pro Lys Val
275 280 285
Ile Glu Asn Ile Arg Asp Gln Thr Ile Ser Tyr Ile Ser Cys Gly Glu
290 295 300
Asn His Thr Ala Leu Ile Thr Asp Ile Gly Leu Met Tyr Thr Phe Gly
305 310 315 320
Asp Gly Arg His Gly Lys Leu Gly Leu Gly Leu Glu Asn Phe Thr Asn
325 330 335
His Phe Ile Pro Thr Leu Cys Ser Asn Phe Leu Arg Phe Ile Val Lys
340 345 350
Leu Val Ala Cys Gly Gly Cys His Met Val Val Phe Ala Ala Pro His
355 360 365
Arg Gly Val Ala Lys Glu Ile Glu Phe Asp Glu Ile Asn Asp Thr Cys
370 375 380
Leu Ser Val Ala Thr Phe Leu Pro Tyr Ser Ser Leu Thr Ser Gly Asn
385 390 395 400
Val Leu Gln Arg Thr Leu Ser Ala Arg Met Arg Arg Arg Glu Arg Glu
405 410 415
Arg Ser Pro Asp Ser Phe Ser Met Arg Arg Thr Leu Pro Pro Ile Glu
420 425 430
Gly Thr Leu Gly Leu Ser Ala Cys Phe Leu Pro Asn Ser Val Phe Pro
435 440 445
Arg Cys Ser Glu Arg Asn Leu Gln Glu Ser Val Leu Ser Glu Gln Asp
450 455 460
Leu Met Gln Pro Glu Glu Pro Asp Tyr Leu Leu Asp Glu Met Thr Lys
465 470 475 480
Glu Ala Glu Ile Asp Asn Ser Ser Thr Val Glu Ser Leu Gly Glu Thr
485 490 495
Thr Asp Ile Leu Asn Met Thr His Ile Met Ser Leu Asn Ser Asn Glu
500 505 510
Lys Ser Leu Lys Leu Ser Pro Val Gln Lys Gln Lys Lys Gln Gln Thr
515 520 525
Ile Gly Glu Leu Thr Gln Asp Thr Ala Leu Thr Glu Asn Asp Asp Ser
530 535 540
Asp Glu Tyr Glu Glu Met Ser Glu Met Lys Glu Gly Lys Ala Cys Lys
545 550 555 560
Gln His Val Ser Gln Gly Ile Phe Met Thr Gln Pro Ala Thr Thr Ile
565 570 575
Glu Ala Phe Ser Asp Glu Glu Val Glu Ile Pro Glu Glu Lys Glu Gly
580 585 590
Ala Glu Asp Ser Lys Gly Asn Gly Ile Glu Glu Gln Glu Val Glu Ala
595 600 605
Asn Glu Glu Asn Val Lys Val His Gly Gly Arg Lys Glu Lys Thr Glu
610 615 620
Ile Leu Ser Asp Asp Leu Thr Asp Lys Ala Glu Asp His Glu Phe Ser
625 630 635 640
Lys Thr Glu Glu Leu Lys Leu Glu Asp Val Asp Glu Glu Ile Asn Ala
645 650 655
Glu Asn Val Glu Ser Lys Lys Lys Thr Val Gly Asp Asp Glu Ser Val
660 665 670
Pro Thr Gly Tyr His Ser Lys Thr Glu Gly Ala Glu Arg Thr Asn Asp
675 680 685
Asp Ser Ser Ala Glu Thr Ile Glu Lys Lys Glu Lys Ala Asn Leu Glu
690 695 700
Glu Arg Ala Ile Cys Glu Tyr Asn Glu Asn Pro Lys Gly Tyr Met Leu
705 710 715 720
Asp Asp Ala Asp Ser Ser Ser Leu Glu Ile Leu Glu Asn Ser Glu Thr
725 730 735
Thr Pro Ser Lys Asp Met Lys Lys Thr Lys Lys Ile Phe Leu Phe Lys
740 745 750
Arg Val Pro Ser Ile Asn Gln Lys Ile Val Lys Asn Asn Asn Glu Pro
755 760 765
Leu Pro Glu Ile Lys Ser Ile Gly Asp Gln Ile Ile Leu Lys Ser Asp
770 775 780
Asn Lys Asp Ala Asp Gln Asn His Met Ser Gln Asn His Gln Asn Ile
785 790 795 800
Pro Pro Thr Asn Thr Glu Arg Arg Ser Lys Ser Cys Thr Ile Leu
805 810 815
<210> 29
<211> 646
<212> PRT
<213> Homo sapiens
<400> 29
Met Arg Glu Pro Glu Glu Leu Met Pro Asp Ser Gly Ala Val Phe Thr
1 5 10 15
Phe Gly Lys Ser Lys Phe Ala Glu Asn Asn Pro Gly Lys Phe Trp Phe
20 25 30
Lys Asn Asp Val Pro Val His Leu Ser Cys Gly Asp Glu His Ser Ala
35 40 45
Val Val Thr Gly Asn Asn Lys Leu Tyr Met Phe Gly Ser Asn Asn Trp
50 55 60
Gly Gln Leu Gly Leu Gly Ser Lys Ser Ala Ile Ser Lys Pro Thr Cys
65 70 75 80
Val Lys Ala Leu Lys Pro Glu Lys Val Lys Leu Ala Ala Cys Gly Arg
85 90 95
Asn His Thr Leu Val Ser Thr Glu Gly Gly Asn Val Tyr Ala Thr Gly
100 105 110
Gly Asn Asn Glu Gly Gln Leu Gly Leu Gly Asp Thr Glu Glu Arg Asn
115 120 125
Thr Phe His Val Ile Ser Phe Phe Thr Ser Glu His Lys Ile Lys Gln
130 135 140
Leu Ser Ala Gly Ser Asn Thr Ser Ala Ala Leu Thr Glu Asp Gly Arg
145 150 155 160
Leu Phe Met Trp Gly Asp Asn Ser Glu Gly Gln Ile Gly Leu Lys Asn
165 170 175
Val Ser Asn Val Cys Val Pro Gln Gln Val Thr Ile Gly Lys Pro Val
180 185 190
Ser Trp Ile Ser Cys Gly Tyr Tyr His Ser Ala Phe Val Thr Thr Asp
195 200 205
Gly Glu Leu Tyr Val Phe Gly Glu Pro Glu Asn Gly Lys Leu Gly Leu
210 215 220
Pro Asn Gln Leu Leu Gly Asn His Arg Thr Pro Gln Leu Val Ser Glu
225 230 235 240
Ile Pro Glu Lys Val Ile Gln Val Ala Cys Gly Gly Glu His Thr Val
245 250 255
Val Leu Thr Glu Asn Ala Val Tyr Thr Phe Gly Leu Gly Gln Phe Gly
260 265 270
Gln Leu Gly Leu Gly Thr Phe Leu Phe Glu Thr Ser Glu Pro Lys Val
275 280 285
Ile Glu Asn Ile Arg Asp Gln Thr Ile Ser Tyr Ile Ser Cys Gly Glu
290 295 300
Asn His Thr Ala Leu Ile Thr Asp Ile Gly Leu Met Tyr Thr Phe Gly
305 310 315 320
Asp Gly Arg His Gly Lys Leu Gly Leu Gly Leu Glu Asn Phe Thr Asn
325 330 335
His Phe Ile Pro Thr Leu Cys Ser Asn Phe Leu Arg Phe Ile Val Lys
340 345 350
Leu Val Ala Cys Gly Gly Cys His Met Val Val Phe Ala Ala Pro His
355 360 365
Arg Gly Val Ala Lys Glu Ile Glu Phe Asp Glu Ile Asn Asp Thr Cys
370 375 380
Leu Ser Val Ala Thr Phe Leu Pro Tyr Ser Ser Leu Thr Ser Gly Asn
385 390 395 400
Val Leu Gln Arg Thr Leu Ser Ala Arg Met Arg Arg Arg Glu Arg Glu
405 410 415
Arg Ser Pro Asp Ser Phe Ser Met Arg Arg Thr Leu Pro Pro Ile Glu
420 425 430
Gly Thr Leu Gly Leu Ser Ala Cys Phe Leu Pro Asn Ser Val Phe Pro
435 440 445
Arg Cys Ser Glu Arg Asn Leu Gln Glu Ser Val Leu Ser Glu Gln Asp
450 455 460
Leu Met Gln Pro Glu Glu Pro Asp Tyr Leu Leu Asp Glu Met Thr Lys
465 470 475 480
Glu Ala Glu Ile Asp Asn Ser Ser Thr Val Glu Ser Leu Gly Glu Thr
485 490 495
Thr Asp Ile Leu Asn Met Thr His Ile Met Ser Leu Asn Ser Asn Glu
500 505 510
Lys Ser Leu Lys Leu Ser Pro Val Gln Lys Gln Lys Lys Gln Gln Thr
515 520 525
Ile Gly Glu Leu Thr Gln Asp Thr Ala Leu Thr Glu Asn Asp Asp Ser
530 535 540
Asp Glu Tyr Glu Glu Met Ser Glu Met Lys Glu Gly Lys Ala Cys Lys
545 550 555 560
Gln His Val Ser Gln Gly Ile Phe Met Thr Gln Pro Ala Thr Thr Ile
565 570 575
Glu Ala Phe Ser Asp Glu Glu Val Glu Ile Pro Glu Glu Lys Glu Gly
580 585 590
Ala Glu Asp Ser Lys Gly Asn Gly Ile Glu Glu Gln Glu Val Glu Ala
595 600 605
Asn Glu Glu Asn Val Lys Val His Gly Gly Arg Lys Glu Lys Thr Glu
610 615 620
Ile Leu Ser Asp Asp Leu Thr Asp Lys Ala Glu Tyr Ser Ala Ser His
625 630 635 640
Ser Gln Ile Val Ser Val
645
<210> 30
<211> 1152
<212> PRT
<213> Homo sapiens
<400> 30
Met Arg Glu Pro Glu Glu Leu Met Pro Asp Ser Gly Ala Val Phe Thr
1 5 10 15
Phe Gly Lys Ser Lys Phe Ala Glu Asn Asn Pro Gly Lys Phe Trp Phe
20 25 30
Lys Asn Asp Val Pro Val His Leu Ser Cys Gly Asp Glu His Ser Ala
35 40 45
Val Val Thr Gly Asn Asn Lys Leu Tyr Met Phe Gly Ser Asn Asn Trp
50 55 60
Gly Gln Leu Gly Leu Gly Ser Lys Ser Ala Ile Ser Lys Pro Thr Cys
65 70 75 80
Val Lys Ala Leu Lys Pro Glu Lys Val Lys Leu Ala Ala Cys Gly Arg
85 90 95
Asn His Thr Leu Val Ser Thr Glu Gly Gly Asn Val Tyr Ala Thr Gly
100 105 110
Gly Asn Asn Glu Gly Gln Leu Gly Leu Gly Asp Thr Glu Glu Arg Asn
115 120 125
Thr Phe His Val Ile Ser Phe Phe Thr Ser Glu His Lys Ile Lys Gln
130 135 140
Leu Ser Ala Gly Ser Asn Thr Ser Ala Ala Leu Thr Glu Asp Gly Arg
145 150 155 160
Leu Phe Met Trp Gly Asp Asn Ser Glu Gly Gln Ile Gly Leu Lys Asn
165 170 175
Val Ser Asn Val Cys Val Pro Gln Gln Val Thr Ile Gly Lys Pro Val
180 185 190
Ser Trp Ile Ser Cys Gly Tyr Tyr His Ser Ala Phe Val Thr Thr Asp
195 200 205
Gly Glu Leu Tyr Val Phe Gly Glu Pro Glu Asn Gly Lys Leu Gly Leu
210 215 220
Pro Asn Gln Leu Leu Gly Asn His Arg Thr Pro Gln Leu Val Ser Glu
225 230 235 240
Ile Pro Glu Lys Val Ile Gln Val Ala Cys Gly Gly Glu His Thr Val
245 250 255
Val Leu Thr Glu Asn Ala Val Tyr Thr Phe Gly Leu Gly Gln Phe Gly
260 265 270
Gln Leu Gly Leu Gly Thr Phe Leu Phe Glu Thr Ser Glu Pro Lys Val
275 280 285
Ile Glu Asn Ile Arg Asp Gln Thr Ile Ser Tyr Ile Ser Cys Gly Glu
290 295 300
Asn His Thr Ala Leu Ile Thr Asp Ile Gly Leu Met Tyr Thr Phe Gly
305 310 315 320
Asp Gly Arg His Gly Lys Leu Gly Leu Gly Leu Glu Asn Phe Thr Asn
325 330 335
His Phe Ile Pro Thr Leu Cys Ser Asn Phe Leu Arg Phe Ile Val Lys
340 345 350
Leu Val Ala Cys Gly Gly Cys His Met Val Val Phe Ala Ala Pro His
355 360 365
Arg Gly Val Ala Lys Glu Ile Glu Phe Asp Glu Ile Asn Asp Thr Cys
370 375 380
Leu Ser Val Ala Thr Phe Leu Pro Tyr Ser Ser Leu Thr Ser Gly Asn
385 390 395 400
Val Leu Gln Arg Thr Leu Ser Ala Arg Met Arg Arg Arg Glu Arg Glu
405 410 415
Arg Ser Pro Asp Ser Phe Ser Met Arg Arg Thr Leu Pro Pro Ile Glu
420 425 430
Gly Thr Leu Gly Leu Ser Ala Cys Phe Leu Pro Asn Ser Val Phe Pro
435 440 445
Arg Cys Ser Glu Arg Asn Leu Gln Glu Ser Val Leu Ser Glu Gln Asp
450 455 460
Leu Met Gln Pro Glu Glu Pro Asp Tyr Leu Leu Asp Glu Met Thr Lys
465 470 475 480
Glu Ala Glu Ile Asp Asn Ser Ser Thr Val Glu Ser Leu Gly Glu Thr
485 490 495
Thr Asp Ile Leu Asn Met Thr His Ile Met Ser Leu Asn Ser Asn Glu
500 505 510
Lys Ser Leu Lys Leu Ser Pro Val Gln Lys Gln Lys Lys Gln Gln Thr
515 520 525
Ile Gly Glu Leu Thr Gln Asp Thr Ala Leu Thr Glu Asn Asp Asp Ser
530 535 540
Asp Glu Tyr Glu Glu Met Ser Glu Met Lys Glu Gly Lys Ala Cys Lys
545 550 555 560
Gln His Val Ser Gln Gly Ile Phe Met Thr Gln Pro Ala Thr Thr Ile
565 570 575
Glu Ala Phe Ser Asp Glu Glu Val Glu Ile Pro Glu Glu Lys Glu Gly
580 585 590
Ala Glu Asp Ser Lys Gly Asn Gly Ile Glu Glu Gln Glu Val Glu Ala
595 600 605
Asn Glu Glu Asn Val Lys Val His Gly Gly Arg Lys Glu Lys Thr Glu
610 615 620
Ile Leu Ser Asp Asp Leu Thr Asp Lys Ala Glu Val Ser Glu Gly Lys
625 630 635 640
Ala Lys Ser Val Gly Glu Ala Glu Asp Gly Pro Glu Gly Arg Gly Asp
645 650 655
Gly Thr Cys Glu Glu Gly Ser Ser Gly Ala Glu His Trp Gln Asp Glu
660 665 670
Glu Arg Glu Lys Gly Glu Lys Asp Lys Gly Arg Gly Glu Met Glu Arg
675 680 685
Pro Gly Glu Gly Glu Lys Glu Leu Ala Glu Lys Glu Glu Trp Lys Lys
690 695 700
Arg Asp Gly Glu Glu Gln Glu Gln Lys Glu Arg Glu Gln Gly His Gln
705 710 715 720
Lys Glu Arg Asn Gln Glu Met Glu Glu Gly Gly Glu Glu Glu His Gly
725 730 735
Glu Gly Glu Glu Glu Glu Gly Asp Arg Glu Glu Glu Glu Glu Lys Glu
740 745 750
Gly Glu Gly Lys Glu Glu Gly Glu Gly Glu Glu Val Glu Gly Glu Arg
755 760 765
Glu Lys Glu Glu Gly Glu Arg Lys Lys Glu Glu Arg Ala Gly Lys Glu
770 775 780
Glu Lys Gly Glu Glu Glu Gly Asp Gln Gly Glu Gly Glu Glu Glu Glu
785 790 795 800
Thr Glu Gly Arg Gly Glu Glu Lys Glu Glu Gly Gly Glu Val Glu Gly
805 810 815
Gly Glu Val Glu Glu Gly Lys Gly Glu Arg Glu Glu Glu Glu Glu Glu
820 825 830
Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu
835 840 845
Gly Glu Glu Glu Glu Gly Glu Gly Lys Gly Glu Glu Glu Gly Glu Glu
850 855 860
Gly Glu Gly Glu Glu Glu Gly Glu Glu Gly Glu Gly Glu Gly Glu Glu
865 870 875 880
Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly Glu
885 890 895
Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly
900 905 910
Glu Glu Glu Glu Gly Glu Gly Lys Gly Glu Glu Glu Gly Glu Glu Gly
915 920 925
Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Gly Glu Asp Gly
930 935 940
Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Trp Glu Gly Glu Glu Glu
945 950 955 960
Glu Gly Glu Gly Glu Gly Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu
965 970 975
Gly Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu Gly Glu Gly Glu Glu
980 985 990
Glu Glu Gly Glu Glu Glu Gly Glu Glu Glu Gly Glu Gly Glu Glu Glu
995 1000 1005
Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu Gly Glu Val Glu Gly Glu
1010 1015 1020
Val Glu Gly Glu Glu Gly Glu Gly Glu Gly Glu Glu Glu Glu Gly Glu
1025 1030 1035 1040
Glu Glu Gly Glu Glu Arg Glu Lys Glu Gly Glu Gly Glu Glu Asn Arg
1045 1050 1055
Arg Asn Arg Glu Glu Glu Glu Glu Glu Glu Gly Lys Tyr Gln Glu Thr
1060 1065 1070
Gly Glu Glu Glu Asn Glu Arg Gln Asp Gly Glu Glu Tyr Lys Lys Val
1075 1080 1085
Ser Lys Ile Lys Gly Ser Val Lys Tyr Gly Lys His Lys Thr Tyr Gln
1090 1095 1100
Lys Lys Ser Val Thr Asn Thr Gln Gly Asn Gly Lys Glu Gln Arg Ser
1105 1110 1115 1120
Lys Met Pro Val Gln Ser Lys Arg Leu Leu Lys Asn Gly Pro Ser Gly
1125 1130 1135
Ser Lys Lys Phe Trp Asn Asn Val Leu Pro His Tyr Leu Glu Leu Lys
1140 1145 1150
<210> 31
<211> 1020
<212> PRT
<213> Homo sapiens
<400> 31
Met Arg Glu Pro Glu Glu Leu Met Pro Asp Ser Gly Ala Val Phe Thr
1 5 10 15
Phe Gly Lys Ser Lys Phe Ala Glu Asn Asn Pro Gly Lys Phe Trp Phe
20 25 30
Lys Asn Asp Val Pro Val His Leu Ser Cys Gly Asp Glu His Ser Ala
35 40 45
Val Val Thr Gly Asn Asn Lys Leu Tyr Met Phe Gly Ser Asn Asn Trp
50 55 60
Gly Gln Leu Gly Leu Gly Ser Lys Ser Ala Ile Ser Lys Pro Thr Cys
65 70 75 80
Val Lys Ala Leu Lys Pro Glu Lys Val Lys Leu Ala Ala Cys Gly Arg
85 90 95
Asn His Thr Leu Val Ser Thr Glu Gly Gly Asn Val Tyr Ala Thr Gly
100 105 110
Gly Asn Asn Glu Gly Gln Leu Gly Leu Gly Asp Thr Glu Glu Arg Asn
115 120 125
Thr Phe His Val Ile Ser Phe Phe Thr Ser Glu His Lys Ile Lys Gln
130 135 140
Leu Ser Ala Gly Ser Asn Thr Ser Ala Ala Leu Thr Glu Asp Gly Arg
145 150 155 160
Leu Phe Met Trp Gly Asp Asn Ser Glu Gly Gln Ile Gly Leu Lys Asn
165 170 175
Val Ser Asn Val Cys Val Pro Gln Gln Val Thr Ile Gly Lys Pro Val
180 185 190
Ser Trp Ile Ser Cys Gly Tyr Tyr His Ser Ala Phe Val Thr Thr Asp
195 200 205
Gly Glu Leu Tyr Val Phe Gly Glu Pro Glu Asn Gly Lys Leu Gly Leu
210 215 220
Pro Asn Gln Leu Leu Gly Asn His Arg Thr Pro Gln Leu Val Ser Glu
225 230 235 240
Ile Pro Glu Lys Val Ile Gln Val Ala Cys Gly Gly Glu His Thr Val
245 250 255
Val Leu Thr Glu Asn Ala Val Tyr Thr Phe Gly Leu Gly Gln Phe Gly
260 265 270
Gln Leu Gly Leu Gly Thr Phe Leu Phe Glu Thr Ser Glu Pro Lys Val
275 280 285
Ile Glu Asn Ile Arg Asp Gln Thr Ile Ser Tyr Ile Ser Cys Gly Glu
290 295 300
Asn His Thr Ala Leu Ile Thr Asp Ile Gly Leu Met Tyr Thr Phe Gly
305 310 315 320
Asp Gly Arg His Gly Lys Leu Gly Leu Gly Leu Glu Asn Phe Thr Asn
325 330 335
His Phe Ile Pro Thr Leu Cys Ser Asn Phe Leu Arg Phe Ile Val Lys
340 345 350
Leu Val Ala Cys Gly Gly Cys His Met Val Val Phe Ala Ala Pro His
355 360 365
Arg Gly Val Ala Lys Glu Ile Glu Phe Asp Glu Ile Asn Asp Thr Cys
370 375 380
Leu Ser Val Ala Thr Phe Leu Pro Tyr Ser Ser Leu Thr Ser Gly Asn
385 390 395 400
Val Leu Gln Arg Thr Leu Ser Ala Arg Met Arg Arg Arg Glu Arg Glu
405 410 415
Arg Ser Pro Asp Ser Phe Ser Met Arg Arg Thr Leu Pro Pro Ile Glu
420 425 430
Gly Thr Leu Gly Leu Ser Ala Cys Phe Leu Pro Asn Ser Val Phe Pro
435 440 445
Arg Cys Ser Glu Arg Asn Leu Gln Glu Ser Val Leu Ser Glu Gln Asp
450 455 460
Leu Met Gln Pro Glu Glu Pro Asp Tyr Leu Leu Asp Glu Met Thr Lys
465 470 475 480
Glu Ala Glu Ile Asp Asn Ser Ser Thr Val Glu Ser Leu Gly Glu Thr
485 490 495
Thr Asp Ile Leu Asn Met Thr His Ile Met Ser Leu Asn Ser Asn Glu
500 505 510
Lys Ser Leu Lys Leu Ser Pro Val Gln Lys Gln Lys Lys Gln Gln Thr
515 520 525
Ile Gly Glu Leu Thr Gln Asp Thr Ala Leu Thr Glu Asn Asp Asp Ser
530 535 540
Asp Glu Tyr Glu Glu Met Ser Glu Met Lys Glu Gly Lys Ala Cys Lys
545 550 555 560
Gln His Val Ser Gln Gly Ile Phe Met Thr Gln Pro Ala Thr Thr Ile
565 570 575
Glu Ala Phe Ser Asp Glu Glu Val Gly Asn Asp Thr Gly Gln Val Gly
580 585 590
Pro Gln Ala Asp Thr Asp Gly Glu Gly Leu Gln Lys Glu Val Tyr Arg
595 600 605
His Glu Asn Asn Asn Gly Val Asp Gln Leu Asp Ala Lys Glu Ile Glu
610 615 620
Lys Glu Ser Asp Gly Gly His Ser Gln Lys Glu Ser Glu Ala Glu Glu
625 630 635 640
Ile Asp Ser Glu Lys Glu Thr Lys Leu Ala Glu Ile Ala Gly Met Lys
645 650 655
Asp Leu Arg Glu Arg Glu Lys Ser Thr Lys Lys Met Ser Pro Phe Phe
660 665 670
Gly Asn Leu Pro Asp Arg Gly Met Asn Thr Glu Ser Glu Glu Asn Lys
675 680 685
Asp Phe Val Lys Lys Arg Glu Ser Cys Lys Gln Asp Val Ile Phe Asp
690 695 700
Ser Glu Arg Glu Ser Val Glu Lys Pro Asp Ser Tyr Met Glu Gly Ala
705 710 715 720
Ser Glu Ser Gln Gln Gly Ile Ala Asp Gly Phe Gln Gln Pro Glu Ala
725 730 735
Ile Glu Phe Ser Ser Gly Glu Lys Glu Asp Asp Glu Val Glu Thr Asp
740 745 750
Gln Asn Ile Arg Tyr Gly Arg Lys Leu Ile Glu Gln Gly Asn Glu Lys
755 760 765
Glu Thr Lys Pro Ile Ile Ser Lys Ser Met Ala Lys Tyr Asp Phe Lys
770 775 780
Cys Asp Arg Leu Ser Glu Ile Pro Glu Glu Lys Glu Gly Ala Glu Asp
785 790 795 800
Ser Lys Gly Asn Gly Ile Glu Glu Gln Glu Val Glu Ala Asn Glu Glu
805 810 815
Asn Val Lys Val His Gly Gly Arg Lys Glu Lys Thr Glu Ile Leu Ser
820 825 830
Asp Asp Leu Thr Asp Lys Ala Glu Asp His Glu Phe Ser Lys Thr Glu
835 840 845
Glu Leu Lys Leu Glu Asp Val Asp Glu Glu Ile Asn Ala Glu Asn Val
850 855 860
Glu Ser Lys Lys Lys Thr Val Gly Asp Asp Glu Ser Val Pro Thr Gly
865 870 875 880
Tyr His Ser Lys Thr Glu Gly Ala Glu Arg Thr Asn Asp Asp Ser Ser
885 890 895
Ala Glu Thr Ile Glu Lys Lys Glu Lys Ala Asn Leu Glu Glu Arg Ala
900 905 910
Ile Cys Glu Tyr Asn Glu Asn Pro Lys Gly Tyr Met Leu Asp Asp Ala
915 920 925
Asp Ser Ser Ser Leu Glu Ile Leu Glu Asn Ser Glu Thr Thr Pro Ser
930 935 940
Lys Asp Met Lys Lys Thr Lys Lys Ile Phe Leu Phe Lys Arg Val Pro
945 950 955 960
Ser Ile Asn Gln Lys Ile Val Lys Asn Asn Asn Glu Pro Leu Pro Glu
965 970 975
Ile Lys Ser Ile Gly Asp Gln Ile Ile Leu Lys Ser Asp Asn Lys Asp
980 985 990
Ala Asp Gln Asn His Met Ser Gln Asn His Gln Asn Ile Pro Pro Thr
995 1000 1005
Asn Thr Glu Arg Arg Ser Lys Ser Cys Thr Ile Leu
1010 1015 1020
<210> 32
<211> 1368
<212> PRT
<213> Streptococcus pyogenes
<400> 32
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 33
<211> 1053
<212> PRT
<213> Staphylococcus aureus
<400> 33
Met Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val
1 5 10 15
Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly
20 25 30
Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg
35 40 45
Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile
50 55 60
Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His
65 70 75 80
Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu
85 90 95
Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu
100 105 110
Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr
115 120 125
Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala
130 135 140
Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys
145 150 155 160
Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr
165 170 175
Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln
180 185 190
Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg
195 200 205
Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys
210 215 220
Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe
225 230 235 240
Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr
245 250 255
Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn
260 265 270
Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe
275 280 285
Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu
290 295 300
Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys
305 310 315 320
Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr
325 330 335
Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala
340 345 350
Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu
355 360 365
Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser
370 375 380
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile
385 390 395 400
Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala
405 410 415
Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln
420 425 430
Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro
435 440 445
Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile
450 455 460
Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg
465 470 475 480
Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys
485 490 495
Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr
500 505 510
Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp
515 520 525
Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu
530 535 540
Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro
545 550 555 560
Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys
565 570 575
Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu
580 585 590
Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile
595 600 605
Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu
610 615 620
Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp
625 630 635 640
Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu
645 650 655
Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys
660 665 670
Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp
675 680 685
Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp
690 695 700
Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys
705 710 715 720
Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys
725 730 735
Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu
740 745 750
Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp
755 760 765
Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile
770 775 780
Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu
785 790 795 800
Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu
805 810 815
Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His
820 825 830
Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly
835 840 845
Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr
850 855 860
Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile
865 870 875 880
Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp
885 890 895
Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr
900 905 910
Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val
915 920 925
Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser
930 935 940
Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala
945 950 955 960
Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly
965 970 975
Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile
980 985 990
Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met
995 1000 1005
Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1010 1015 1020
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu
1025 1030 1035 1040
Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1045 1050
<210> 34
<211> 1300
<212> PRT
<213> Francisella tularensis
<400> 34
Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys
20 25 30
Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys
35 40 45
Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu
50 55 60
Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser
65 70 75 80
Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys
85 90 95
Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr
100 105 110
Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile
115 120 125
Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln
130 135 140
Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr
145 150 155 160
Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr
165 170 175
Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser
180 185 190
Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu
195 200 205
Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys
210 215 220
Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu
225 230 235 240
Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg
245 250 255
Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr
260 265 270
Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys
275 280 285
Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile
290 295 300
Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys
305 310 315 320
Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser
325 330 335
Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met
340 345 350
Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys
355 360 365
Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln
370 375 380
Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr
385 390 395 400
Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala
405 410 415
Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn
420 425 430
Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala
435 440 445
Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn
450 455 460
Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala
465 470 475 480
Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys
485 490 495
Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys
500 505 510
Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp
515 520 525
Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His
530 535 540
Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His
545 550 555 560
Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val
565 570 575
Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser
580 585 590
Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly
595 600 605
Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys
610 615 620
Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile
625 630 635 640
Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys
645 650 655
Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val
660 665 670
Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile
675 680 685
Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln
690 695 700
Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe
705 710 715 720
Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp
725 730 735
Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu
740 745 750
Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn
755 760 765
Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr
770 775 780
Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg
785 790 795 800
Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn
805 810 815
Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr
820 825 830
Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala
835 840 845
Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu
850 855 860
Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe
865 870 875 880
His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe
885 890 895
Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His
900 905 910
Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu
915 920 925
Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile
930 935 940
Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile
945 950 955 960
Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn
965 970 975
Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile
980 985 990
Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu
995 1000 1005
Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr
1010 1015 1020
Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe
1025 1030 1035 1040
Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln
1045 1050 1055
Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly
1060 1065 1070
Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val
1075 1080 1085
Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys
1090 1095 1100
Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp
1105 1110 1115 1120
Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys
1125 1130 1135
Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile
1140 1145 1150
Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val
1155 1160 1165
Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu
1170 1175 1180
Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp
1185 1190 1195 1200
Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln
1205 1210 1215
Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro
1220 1225 1230
Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys
1235 1240 1245
Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu
1250 1255 1260
Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys
1265 1270 1275 1280
Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln
1285 1290 1295
Asn Arg Asn Asn
1300
<210> 35
<211> 297
<212> PRT
<213> Adeno-associated virus-1
<400> 35
Thr Phe Ser Tyr Thr Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala
1 5 10 15
His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr
20 25 30
Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn
35 40 45
Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln
50 55 60
Pro Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
65 70 75 80
Lys Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
85 90 95
Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr
100 105 110
Ala Met Ala Ser His Lys Asp Asp Glu Asp Lys Phe Phe Pro Met Ser
115 120 125
Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala
130 135 140
Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn Phe Lys Asp
165 170 175
Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly Lys
180 185 190
Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile Thr
195 200 205
Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg Phe
210 215 220
Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala Thr
225 230 235 240
Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp
245 250 255
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
260 265 270
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
275 280 285
Asn Pro Pro Pro Gln Ile Leu Ile Lys
290 295
<210> 36
<211> 240
<212> PRT
<213> Adeno-associated virus-6
<400> 36
Thr Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala
1 5 10 15
His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr
20 25 30
Leu Tyr Tyr Leu Asn Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn
35 40 45
Lys Asp Leu Leu Phe Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln
50 55 60
Pro Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
65 70 75 80
Lys Thr Lys Thr Asp Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
85 90 95
Ser Lys Tyr Asn Leu Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr
100 105 110
Ala Met Ala Ser His Lys Asp Asp Lys Asp Lys Phe Phe Pro Met Ser
115 120 125
Gly Val Met Ile Phe Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala
130 135 140
Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Arg Phe Gly Thr Val Ala Val Asn Leu Gln Ser
165 170 175
Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His Val Met Gly Ala Leu
180 185 190
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
195 200 205
Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu
210 215 220
Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys
225 230 235 240
<210> 37
<211> 240
<212> PRT
<213> Adeno-associated virus-3
<400> 37
Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His
1 5 10 15
Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu
20 25 30
Tyr Tyr Leu Asn Arg Thr Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln
35 40 45
Ser Arg Leu Leu Phe Ser Gln Ala Gly Pro Gln Ser Met Ser Leu Gln
50 55 60
Ala Arg Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser
65 70 75 80
Lys Thr Ala Asn Asp Asn Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala
85 90 95
Ser Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro
100 105 110
Ala Met Ala Ser His Lys Asp Asp Glu Glu Lys Phe Phe Pro Met His
115 120 125
Gly Asn Leu Ile Phe Gly Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu
130 135 140
Leu Asp Asn Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Gln Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser
165 170 175
Ser Asn Thr Ala Pro Thr Thr Gly Thr Val Asn His Gln Gly Ala Leu
180 185 190
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
195 200 205
Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu
210 215 220
Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Met Ile Lys
225 230 235 240
<210> 38
<211> 240
<212> PRT
<213> Adeno-associated virus-2
<400> 38
Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His
1 5 10 15
Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu
20 25 30
Tyr Tyr Leu Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser
35 40 45
Arg Leu Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser
50 55 60
Arg Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys
65 70 75 80
Thr Ser Ala Asp Asn Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr
85 90 95
Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala
100 105 110
Met Ala Ser His Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly
115 120 125
Val Leu Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile
130 135 140
Glu Lys Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro
145 150 155 160
Val Ala Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly
165 170 175
Asn Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln Gly Val Leu Pro
180 185 190
Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp
195 200 205
Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro Ser Pro Leu Met
210 215 220
Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn
225 230 235 240
<210> 39
<211> 243
<212> PRT
<213> Adeno-associated virus-8
<400> 39
Asn Phe Gln Phe Thr Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser
1 5 10 15
Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp
20 25 30
Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala
35 40 45
Asn Thr Gln Thr Leu Gly Phe Ser Gln Gly Gly Pro Asn Thr Met Ala
50 55 60
Asn Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg
65 70 75 80
Val Ser Thr Thr Thr Gly Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
85 90 95
Ala Gly Thr Lys Tyr His Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro
100 105 110
Gly Ile Ala Met Ala Thr His Lys Asp Asp Glu Glu Arg Phe Phe Pro
115 120 125
Ser Asn Gly Ile Leu Ile Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn
130 135 140
Ala Asp Tyr Ser Asp Val Met Leu Thr Ser Glu Glu Glu Ile Lys Thr
145 150 155 160
Thr Asn Pro Val Ala Thr Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu
165 170 175
Gln Gln Gln Asn Thr Ala Pro Gln Ile Gly Thr Val Asn Ser Gln Gly
180 185 190
Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly
195 200 205
Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser
210 215 220
Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu
225 230 235 240
Ile Lys Asn
<210> 40
<211> 243
<212> PRT
<213> Adeno-associated virus-8.1
<400> 40
Asn Phe Gln Phe Thr Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser
1 5 10 15
Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp
20 25 30
Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala
35 40 45
Asn Thr Gln Thr Leu Gly Phe Ser Gln Gly Gly Pro Asn Thr Met Ala
50 55 60
Asn Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg
65 70 75 80
Val Ser Thr Thr Thr Gly Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
85 90 95
Ala Gly Thr Lys Tyr His Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro
100 105 110
Gly Ile Ala Met Ala Thr His Lys Asp Asp Glu Glu Arg Phe Phe Pro
115 120 125
Ser Asn Gly Ile Leu Ile Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn
130 135 140
Ala Asp Tyr Ser Asp Val Met Leu Thr Ser Glu Glu Glu Ile Lys Thr
145 150 155 160
Thr Asn Pro Val Ala Thr Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu
165 170 175
Gln Gly Gln Arg Gln Ala Ala Gln Ile Gly Thr Val Asn Ser Gln Gly
180 185 190
Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly
195 200 205
Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser
210 215 220
Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu
225 230 235 240
Ile Lys Asn
<210> 41
<211> 241
<212> PRT
<213> Adeno-associated virus-8 Rh8
<400> 41
Phe Gln Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr
1 5 10 15
Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln
20 25 30
Tyr Leu Tyr Tyr Leu Val Arg Thr Gln Thr Thr Gly Thr Gly Gly Thr
35 40 45
Gln Thr Leu Ala Phe Ser Gln Ala Gly Pro Ser Ser Met Ala Asn Gln
50 55 60
Ala Arg Asn Trp Val Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser
65 70 75 80
Thr Thr Thr Asn Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala
85 90 95
Ala Lys Phe Lys Leu Asn Gly Arg Asp Ser Leu Met Asn Pro Gly Val
100 105 110
Ala Met Ala Ser His Lys Asp Asp Asp Asp Arg Phe Phe Pro Ser Ser
115 120 125
Gly Val Leu Ile Phe Gly Lys Gln Gly Ala Gly Asn Asp Gly Val Asp
130 135 140
Tyr Ser Gln Val Leu Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Glu Tyr Gly Ala Val Ala Ile Asn Asn Gln Ala
165 170 175
Ala Asn Thr Gln Ala Gln Thr Gly Leu Val His Asn Gln Gly Val Ile
180 185 190
Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile
195 200 205
Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu
210 215 220
Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys
225 230 235 240
Asn
<210> 42
<211> 243
<212> PRT
<213> Adeno-associated virus-10
<400> 42
Asn Phe Glu Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser
1 5 10 15
Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp
20 25 30
Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Gln Ser Thr Gly Gly Thr Gln
35 40 45
Gly Thr Gln Gln Leu Leu Phe Ser Gln Ala Gly Pro Ala Asn Met Ser
50 55 60
Ala Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg
65 70 75 80
Val Ser Thr Thr Leu Ser Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
85 90 95
Gly Ala Thr Lys Tyr His Leu Asn Gly Arg Asp Ser Leu Val Asn Pro
100 105 110
Gly Val Ala Met Ala Thr His Lys Asp Asp Glu Glu Arg Phe Phe Pro
115 120 125
Ser Ser Gly Val Leu Met Phe Gly Lys Gln Gly Ala Gly Arg Asp Asn
130 135 140
Val Asp Tyr Ser Ser Val Met Leu Thr Ser Glu Glu Glu Ile Lys Thr
145 150 155 160
Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly Val Val Ala Asp Asn Leu
165 170 175
Gln Gln Ala Asn Thr Gly Pro Ile Val Gly Asn Val Asn Ser Gln Gly
180 185 190
Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly
195 200 205
Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser
210 215 220
Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu
225 230 235 240
Ile Lys Asn
<210> 43
<211> 242
<212> PRT
<213> Adeno-associated virus-7
<400> 43
Phe Glu Phe Ser Tyr Ser Phe Glu Asp Val Pro Phe His Ser Ser Tyr
1 5 10 15
Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln
20 25 30
Tyr Leu Tyr Tyr Leu Ala Arg Thr Gln Ser Asn Pro Gly Gly Thr Ala
35 40 45
Gly Asn Arg Glu Leu Gln Phe Tyr Gln Gly Gly Pro Ser Thr Met Ala
50 55 60
Glu Gln Ala Lys Asn Trp Leu Pro Gly Pro Cys Phe Arg Gln Gln Arg
65 70 75 80
Val Ser Lys Thr Leu Asp Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr
85 90 95
Gly Ala Thr Lys Tyr His Leu Asn Gly Arg Asn Ser Leu Val Asn Pro
100 105 110
Gly Val Ala Met Ala Thr His Lys Asp Asp Glu Asp Arg Phe Phe Pro
115 120 125
Ser Ser Gly Val Leu Ile Phe Gly Lys Thr Gly Ala Thr Asn Lys Thr
130 135 140
Thr Leu Glu Asn Val Leu Met Thr Asn Glu Glu Glu Ile Arg Pro Thr
145 150 155 160
Asn Pro Val Ala Thr Glu Glu Tyr Gly Ile Val Ser Ser Asn Leu Gln
165 170 175
Ala Ala Asn Thr Ala Ala Gln Thr Gln Val Val Asn Asn Gln Gly Ala
180 185 190
Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro
195 200 205
Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro
210 215 220
Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile
225 230 235 240
Lys Asn
<210> 44
<211> 240
<212> PRT
<213> Adeno-associated virus-9
<400> 44
Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser Tyr
1 5 10 15
Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln
20 25 30
Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln
35 40 45
Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val Gln
50 55 60
Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser
65 70 75 80
Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala
85 90 95
Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly Pro
100 105 110
Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser
115 120 125
Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val Asp
130 135 140
Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln Ser
165 170 175
Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu
180 185 190
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
195 200 205
Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu
210 215 220
Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile Lys
225 230 235 240
<210> 45
<211> 240
<212> PRT
<213> Adeno-associated virus-9.1
<400> 45
Phe Gln Phe Ser Tyr Glu Phe Glu Asn Val Pro Phe His Ser Ser Tyr
1 5 10 15
Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln
20 25 30
Tyr Leu Tyr Tyr Leu Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln
35 40 45
Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn Met Ala Val Gln
50 55 60
Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser
65 70 75 80
Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala
85 90 95
Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met Asn Pro Gly Pro
100 105 110
Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser
115 120 125
Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg Asp Asn Val Asp
130 135 140
Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn
145 150 155 160
Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr Asn His Gln Ser
165 170 175
Gly Gln Ala Gln Ala Ala Thr Gly Trp Val Gln Asn Gln Gly Ile Leu
180 185 190
Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
195 200 205
Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu
210 215 220
Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile Lys
225 230 235 240
<210> 46
<211> 240
<212> PRT
<213> Adeno-associated virus-5
<400> 46
Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser
1 5 10 15
Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp
20 25 30
Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln
35 40 45
Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp
50 55 60
Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly
65 70 75 80
Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu
85 90 95
Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr
100 105 110
Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile
115 120 125
Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu
130 135 140
Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg
145 150 155 160
Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser
165 170 175
Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro
180 185 190
Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp
195 200 205
Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met
210 215 220
Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn
225 230 235 240
<210> 47
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 47
Leu Ala Lys Asp Ala Thr Lys Asn Ala
1 5
<210> 48
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 48
Pro Ala His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 49
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 49
Leu Ala His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 50
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 50
Leu Ala Thr Thr Ser Gln Asn Lys Pro Ala
1 5 10
<210> 51
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 51
Leu Ala Ile Ser Asp Gln Thr Lys His Ala
1 5 10
<210> 52
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 52
Ile Ala Arg Gly Val Ala Pro Ser Ser Ala
1 5 10
<210> 53
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 53
Leu Ala Pro Asp Ser Thr Thr Arg Ser Ala
1 5 10
<210> 54
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 54
Leu Ala Lys Gly Thr Glu Leu Lys Pro Ala
1 5 10
<210> 55
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 55
Leu Ala Ile Ile Asp Ala Thr Lys Asn Ala
1 5 10
<210> 56
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 56
Leu Ala Val Asp Gly Ala Gln Arg Ser Ala
1 5 10
<210> 57
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 57
Pro Ala Pro Gln Asp Thr Thr Lys Lys Ala
1 5 10
<210> 58
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 58
Leu Pro His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 59
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 59
Leu Ala Lys Asp Ala Thr Lys Thr Ile Ala
1 5 10
<210> 60
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 60
Leu Ala Lys Gln Gln Ser Ala Ser Thr Ala
1 5 10
<210> 61
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 61
Leu Ala Lys Ser Asp Gln Ser Lys Pro Ala
1 5 10
<210> 62
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 62
Leu Ser His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 63
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 63
Leu Ala Ala Asn Gln Pro Ser Lys Pro Ala
1 5 10
<210> 64
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 64
Leu Ala Val Ser Asp Ser Thr Lys Ala Ala
1 5 10
<210> 65
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 65
Leu Ala Ala Gln Gly Thr Ala Lys Pro Ala
1 5 10
<210> 66
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 66
Leu Ala Pro Asp Gln Thr Thr Arg Asn Ala
1 5 10
<210> 67
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 67
Leu Ala Ala Ser Asp Ser Thr Lys Ala Ala
1 5 10
<210> 68
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 68
Leu Ala Pro Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 69
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 69
Leu Ala Lys Ala Asp Glu Thr Arg Pro Ala
1 5 10
<210> 70
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 70
Leu Ala His Gln Asp Thr Ala Lys Asn Ala
1 5 10
<210> 71
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 71
Leu Ala His Gln Asp Thr Lys Lys Asn Ala
1 5 10
<210> 72
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 72
Leu Ala His Gln Asp Thr Thr Lys His Ala
1 5 10
<210> 73
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 73
Leu Ala His Gln Asp Thr Thr Lys Lys Ala
1 5 10
<210> 74
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 74
Leu Ala His Gln Asp Thr Thr Arg Asn Ala
1 5 10
<210> 75
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 75
Leu Ala His Gln Asp Thr Thr Thr Asn Ala
1 5 10
<210> 76
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 76
Leu Ala His Gln Gly Thr Thr Lys Asn Ala
1 5 10
<210> 77
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 77
Leu Ala His Gln Val Thr Thr Lys Asn Ala
1 5 10
<210> 78
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 78
Leu Ala Ile Ser Asp Gln Ser Lys Pro Ala
1 5 10
<210> 79
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 79
Leu Ala Lys Asp Ala Thr Lys Thr Ala
1 5
<210> 80
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 80
Leu Ala Lys Asp Thr Thr Lys Asn Ala
1 5
<210> 81
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 81
Leu Ala Lys Ser Asp Gln Ser Arg Pro Ala
1 5 10
<210> 82
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 82
Leu Ala Pro Gln Asp Thr Lys Lys Asn Ala
1 5 10
<210> 83
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 83
Leu Ala Thr Ser Asp Ser Thr Lys Ala Ala
1 5 10
<210> 84
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 84
Leu Ala Val Asp Gly Ser Gln Arg Ser Ala
1 5 10
<210> 85
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 85
Leu Pro Ile Ser Asp Gln Thr Lys His Ala
1 5 10
<210> 86
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 86
Leu Pro Lys Asp Ala Thr Lys Thr Ile Ala
1 5 10
<210> 87
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 87
Leu Pro Pro Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 88
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 88
Pro Ala Pro Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 89
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 89
Gln Ala His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 90
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 90
Leu Ala His Glu Thr Ser Pro Arg Pro Ala
1 5 10
<210> 91
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 91
Leu Ala Lys Ser Thr Ser Thr Ala Pro Ala
1 5 10
<210> 92
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 92
Leu Ala Asp Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 93
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 93
Leu Ala Glu Ser Asp Gln Ser Lys Pro Ala
1 5 10
<210> 94
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 94
Leu Ala His Lys Asp Thr Thr Lys Asn Ala
1 5 10
<210> 95
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 95
Leu Ala His Lys Thr Gln Gln Lys Met
1 5
<210> 96
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 96
Leu Ala His Gln Asp Thr Thr Glu Asn Ala
1 5 10
<210> 97
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 97
Leu Ala His Gln Asp Thr Thr Ile Asn Ala
1 5 10
<210> 98
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 98
Leu Ala His Gln Asp Thr Thr Lys Lys Thr
1 5 10
<210> 99
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 99
Leu Ala His Gln Asp Thr Thr Lys Asn Asp
1 5 10
<210> 100
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 100
Leu Ala His Gln Asp Thr Thr Lys Asn Thr
1 5 10
<210> 101
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 101
Leu Ala His Gln Asp Thr Thr Lys Asn Val
1 5 10
<210> 102
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 102
Leu Ala His Gln Asp Thr Thr Lys Thr Met
1 5 10
<210> 103
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 103
Leu Ala His Gln Asn Thr Thr Lys Asn Ala
1 5 10
<210> 104
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 104
Leu Ala His Arg Asp Thr Thr Lys Asn Ala
1 5 10
<210> 105
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 105
Leu Ala Ile Ser Asp Gln Thr Asn His Ala
1 5 10
<210> 106
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 106
Leu Ala Lys Gln Lys Ser Ala Ser Thr Ala
1 5 10
<210> 107
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 107
Leu Ala Lys Ser Asp Gln Cys Lys Pro Ala
1 5 10
<210> 108
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 108
Leu Ala Lys Ser Asp Gln Ser Lys Pro Asp
1 5 10
<210> 109
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 109
Leu Ala Lys Ser Asp Gln Ser Asn Pro Ala
1 5 10
<210> 110
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 110
Leu Ala Lys Ser Tyr Gln Ser Lys Pro Ala
1 5 10
<210> 111
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 111
Leu Ala Asn Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 112
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 112
Leu Ala Pro Gln Asn Thr Thr Lys Asn Ala
1 5 10
<210> 113
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 113
Leu Ala Pro Ser Ser Ile Gln Lys Pro Ala
1 5 10
<210> 114
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 114
Leu Ala Gln Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 115
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 115
Leu Ala Tyr Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 116
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 116
Leu Asp His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 117
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 117
Leu Asp His Gln Asp Thr Thr Lys Ser Ala
1 5 10
<210> 118
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 118
Leu Gly His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 119
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 119
Leu Pro His Gln Asp Thr Thr Lys Asn Asp
1 5 10
<210> 120
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 120
Leu Pro His Gln Asp Thr Thr Lys Asn Thr
1 5 10
<210> 121
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 121
Leu Pro His Gln Asp Thr Thr Asn Asn Ala
1 5 10
<210> 122
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 122
Leu Thr His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 123
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 123
Leu Thr Lys Asp Ala Thr Lys Thr Ile Ala
1 5 10
<210> 124
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 124
Leu Thr Pro Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 125
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 125
Leu Val His Gln Asp Thr Thr Lys Asn Ala
1 5 10
<210> 126
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 126
Leu Ala Lys Ala Asn Gln Asn Thr Pro Ala
1 5 10
<210> 127
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 127
Leu Ala Thr Thr Pro Ile Thr Lys Pro Ala
1 5 10
<210> 128
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 128
Leu Ala Thr Thr Pro Ile Ala Lys Pro Ala
1 5 10
<210> 129
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 129
Leu Ala Ile Glu Asp His Thr Lys Ser Ala
1 5 10
<210> 130
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 130
Leu Ala Gln Ser Glu His Gln Arg Pro Ala
1 5 10
<210> 131
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 131
Leu Ala Lys Ser Pro Asn Lys Asp Asn Ala
1 5 10
<210> 132
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 132
Leu Ala Asn Gln Asp Tyr Thr Lys Thr Ala
1 5 10
<210> 133
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 133
Leu Ala Asn Ser Thr Asp Gln Thr Arg Ala
1 5 10
<210> 134
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 134
Leu Ala Leu Gly Glu Thr Thr Arg Pro Ala
1 5 10
<210> 135
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 135
Leu Ala Asn Ser Thr Glu Gln Thr Arg Ala
1 5 10
<210> 136
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 136
Leu Ala Gln Ala Asp Thr Thr Lys Asn Ala
1 5 10
<210> 137
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 137
Leu Ala Ser Lys Asp Ile Thr Lys Thr Ala
1 5 10
<210> 138
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 138
Leu Ala Ser Pro Arg His Asn Lys Lys Cys
1 5 10
<210> 139
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 139
Leu Ala His Gln Asp Thr Thr Lys Thr Ile Ala
1 5 10
<210> 140
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 140
Leu Ala Ala Gln Gly Thr Ala Asn Leu
1 5
<210> 141
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 141
Val Ala Ile Glu Asp His Thr Lys Ser Ala
1 5 10
<210> 142
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 142
Leu Ala Lys Ala Asn Gln Asn Thr Pro Lys Asn Ala
1 5 10
<210> 143
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<220>
<221> MISC_FEATURE
<222> (10)
<223> Xaa is any amino acid
<400> 143
Leu Ala His Gln Asp Thr Thr Lys Lys Xaa
1 5 10
<210> 144
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 144
Leu Ala His Gln Asp Thr Thr Lys Lys Asp
1 5 10
<210> 145
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 145
Leu Ala His Gln Asp Thr Thr Lys Lys Val
1 5 10
<210> 146
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 146
Leu Ala His Gln Asp Thr Thr Lys Lys Met
1 5 10
<210> 147
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<400> 147
Leu Gly Glu Thr Thr Arg Pro
1 5
<210> 148
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<220>
<221> MISC_FEATURE
<222> (9)
<223> Xaa is Lys, Thr, Asn, or His
<220>
<221> MISC_FEATURE
<222> (10)
<223> Xaa is Ala, Thr, Val, Ile, Met, or Asp
<220>
<221> MISC_FEATURE
<222> (11)
<223> Xaa is Ala
<400> 148
Leu Ala His Gln Asp Thr Thr Lys Xaa Xaa Xaa
1 5 10
<210> 149
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<220>
<221> MISC_FEATURE
<222> (3)
<223> Xaa is Ala, Pro, Asp, or His
<220>
<221> MISC_FEATURE
<222> (5)
<223> Xaa is Gly or Asp
<220>
<221> MISC_FEATURE
<222> (7)
<223> Xaa is Ala, Thr, or Lys
<220>
<221> MISC_FEATURE
<222> (8)
<223> Xaa is Asn, Glu, Lys, Arg, or Thr
<220>
<221> MISC_FEATURE
<222> (9)
<223> Xaa is Leu, Asn, Lys, or Thr
<220>
<221> MISC_FEATURE
<222> (10)
<223> Xaa is Ala, Thr, Asp, Val, or Met
<400> 149
Leu Ala Xaa Gln Xaa Thr Xaa Xaa Xaa Xaa
1 5 10
<210> 150
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is Val or Leu
<220>
<221> MISC_FEATURE
<222> (3)
<223> Xaa is Ile, Val, His, or Asp
<220>
<221> MISC_FEATURE
<222> (4)
<223> Xaa is Glu, Ser, Lys, or Gln
<220>
<221> MISC_FEATURE
<222> (6)
<223> Xaa is His, Ser, or Thr
<220>
<221> MISC_FEATURE
<222> (9)
<223> Xaa is Ser, Ala, Asn, His, or Lys
<400> 150
Xaa Ala Xaa Xaa Asp Xaa Thr Lys Xaa Ala
1 5 10
<210> 151
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic peptide
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is Leu
<220>
<221> MISC_FEATURE
<222> (2)
<223> Xaa is Ala
<220>
<221> MISC_FEATURE
<222> (3)
<223> Xaa is Lys, Leu, or Pro
<220>
<221> MISC_FEATURE
<222> (5)
<223> Xaa is Asn, His, Pro, or Tyr
<220>
<221> MISC_FEATURE
<222> (7)
<223> Xaa is Asn, Gly, Val, or Asp
<220>
<221> MISC_FEATURE
<222> (9)
<223> Xaa is Pro or Thr
<400> 151
Xaa Xaa Xaa Ala Xaa Gln Xaa Thr Xaa Lys Asn Ala
1 5 10
<210> 152
<211> 27
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic sequence
<400> 152
cgcaaucagu gaaugcuuau acauccg 27
Claims (33)
- 하기를 포함하는, 재조합 아데노-관련된 바이러스 (rAAV) 비리온:
a) 변이체 AAV 캡시드 단백질로서, 상기 변이체 AAV 캡시드 단백질은 상응하는 친계 AAV 캡시드 단백질에 대해 상기 캡시드 단백질 GH 루프에서 약 5개의 아미노산 내지 약 20개의 아미노산의 길이를 갖는 이종성 펩타이드의 삽입을 포함하고, 그리고 상기 변이체 캡시드 단백질은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 대조군 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 증가된 감염성을 부여하는, 상기 변이체 AAV 캡시드 단백질; 및
b) 이종성 유전자 산물을 인코딩하는 뉴클레오타이드 서열을 포함하는 이종성 핵산. - 청구항 1에 있어서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 대조군 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 적어도 5-배수 증가된 감염성을 나타내는, rAAV 비리온.
- 청구항 1에 있어서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해 상기 망막 세포의 감염성과 비교된 망막 세포의 적어도 10-배수 증가된 감염성을 나타내는, rAAV 비리온.
- 청구항 1에 있어서, 상기 rAAV 비리온은 상기 상응하는 친계 AAV 캡시드 단백질을 포함하는 AAV 비리온에 의해 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 또는 망막 색소 상피에 대한 국제화의 정도와 비교하여, 내부 핵 층, 외부 핵 층, 광수용체 층, 신경절 세포 층, 및 망막 색소 상피 중 하나 이상에 대해 적어도 5-배수 증가된 국재화를 나타내는, rAAV 비리온.
- 청구항 1에 있어서, 상기 삽입 부위는 AAV2의 VP1의 아미노산 570 및 611에 상응하는 아미노산, 또는 또 다른 AAV 혈청형의 캡시드 단백질 내의 상응하는 위치 사이에 있는, rAAV 비리온.
- 청구항 4에 있어서, 상기 삽입 부위는 AAV2의 VP1의 아미노산 587 내지 588에 상응하는 아미노산, 또는 또 다른 AAV 혈청형의 캡시드 단백질 내의 상응하는 위치 사이에 있는, rAAV 비리온.
- 청구항 1 내지 6 중 어느 한 항에 있어서, 유전자 산물은 간섭 RNA 또는 압타머인, rAAV 비리온.
- 청구항 1 내지 6 중 어느 한 항에 있어서, 상기 유전자 산물은 폴리펩타이드인, rAAV 비리온.
- 청구항 8에 있어서, 상기 폴리펩타이드는 신경보호성 폴리펩타이드, 항-혈관신생 폴리펩타이드, 또는 망막 세포의 기능을 향상시키는 폴리펩타이드인, rAAV 비리온.
- 청구항 8에 있어서, 상기 폴리펩타이드는 RNA-유도된 엔도뉴클레아제인, rAAV 비리온.
- 청구항 10에 있어서, 상기 RNA-유도된 엔도뉴클레아제는 Cas9 폴리펩타이드인, rAAV 비리온.
- 청구항 10에 있어서, 상기 유전자 산물은 RNA-유도된 엔도뉴클레아제 및 가이드 RNA인, rAAV 비리온.
- 청구항 1 내지 12 중 어느 한 항에 있어서, 상기 이종성 펩타이드는 식 I-X 중 임의의 하나의 것인, rAAV 비리온.
- 청구항 1 내지 12 중 어느 한 항에 있어서, 상기 이종성 펩타이드는 하기 식 I의 펩타이드인, rAAV 비리온:
X1X2X3X4X5X6X7X8X9X10,
식 중:
X1는 Leu, Ile, Pro, 또는 Gln이고;
X2는 Ala, Pro, Ser, Asp, Gly, Thr, 또는 Val이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, 또는 Tyr이고;
X4는, 존재한다면, Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, 또는 Arg이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, 또는 Tyr이고;
X6는 Thr, Ala, Gln, Ser, Glu, Pro, 또는 Ile;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, 또는 Cys이고;
X8는 Lys, Ser, Arg, Thr, Ala, Glu, Ile, 또는 Asn이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala, Phe, Asp, Thr, Val, 또는 Met이다. - 청구항 14에 있어서, 상기 이종성 펩타이드는 하기 아미노산 서열 중 하나를 포함하는, rAAV 비리온: (1) LAKDATKNA; (2) PAHQDTTKNA; (3) LAHQDTTKNA; (4) LATTSQNKPA; (5) LAISDQTKHA; (6) IARGVAPSSA; (7) LAPDSTTRSA; (8) LAKGTELKPA; (9) LAIIDATKNA; (10) LAVDGAQRSA; (11) PAPQDTTKKA; (12) LPHQDTTKNA; (13) LAKDATKTIA; (14) LAKQQSASTA; (15) LAKSDQSKPA; (16) LSHQDTTKNA; (17) LAANQPSKPA; (18) LAVSDSTKAA; (19) LAAQGTAKKPA; (20) LAPDQTTRNA; (21) LAASDSTKAA; (22) LAPQDTTKNA; (23) LAKADETRPA; (24) LAHQDTAKNA; (25) LAHQDTKKNA; (26) LAHQDTTKHA; (27) LAHQDTTKKA; (28) LAHQDTTRNA; (29) LAHQDTTNA; (30) LAHQGTTKNA; (31) LAHQVTTKNA; (32) LAISDQSKPA; (33) LADATKTA; (34) LAKDTTKNA; (35) LAKSDQSRPA; (36) LAPQDTKKNA; (37) LATSDSTKAA; (38) LAVDGSQRSA; (39) LPISDQTKHA; (40) LPKDATKTIA; (41) LPPQDTTKNA; (42) PAPQDTTKNA; (43) QAHQDTTKNA; (44) LAHETSPRPA; (45) LAKSTSTAPA; (46) LADQDTTKNA; (47) LAESDQSKPA; (48) LAHKDTTKNA; (49) LAHKTQQKM; (50) LAHQDTTENA; (51) LAHQDTTINA; (52) LAHQDTTKKT; (53) LAHQDTTKND; (54) LAHQDTTKNT; (55) LAHQDTTKNV; (56) LAHQDTTKTM; (57) LAHQNTTKNA; (58) LAHRDTTKNA; (59) LAISDQTNHA; (60) LAKQKSASTA; (61) LAKSDQCKPA; (62) LAKSDQSKPD; (63) LAKSDQSNPA; (64) LAKSYQSKPA; (65) LANQDTTKNA; (66) LAPQNTTKNA; (67) LAPSSIQKPA; (68) LAQQDTTKNA; (69) LAYQDTTKNA; (70) LDHQDTTKNA; (71) LDHQDTTKSA; (72) LGHQDTTKNA; (73) LPHQDTTKND; (74) LPHQDTTKNT; (75) LPHQDTTNNA; (76) LTHQDTTKNA; (77) LTKDATKTIA; (78) LTPQDTTKNA; 및 (79) LVHQDTTKNA.
- 청구항 1 내지 12 중 어느 한 항에 있어서, 상기 이종성 펩타이드는 하기 식 II의 펩타이드인, rAAV 비리온:
X1X2X3X4X5X6X7X8X9X10,
X1는 Leu, Ile, 또는 Pro이고;
X2는 Ala, Pro, 또는 Ser이고;
X3는 Lys, His, Thr, Ile, Pro, Val, Arg, 또는 Ala이고;
X4 (존재하면)는 Gln, Asp, Ser, Gly, Thr, Ile, 또는 Asn이고;
X5는 Asp, Ser, Gln, Val, Thr, Gly, 또는 Ala이고;
X6은 Thr, Ala, Gln, Ser, Glu, 또는 Pro이고;
X7는 Thr, Ser, Asn, Pro, Leu, Gln, Lys, 또는 Ala이고;
X8는 Lys, Ser, Arg, 또는 Thr이고;
X9는 Asn, Pro, Ser, Lys, His, Ile, Thr, 또는 Ala이고; 그리고
X10는 Ala이다. - 청구항 16에 있어서, 상기 펩타이드는 하기 아미노산 서열 중 하나를 포함하는, rAAV 비리온: (1) LAKDATKNA; (2) PAHQDTTKNA; (3) LAHQDTTKNA; (4) LATTSQNKPA; (5) LAISDQTKHA; (6) IARGVAPSSA; (7) LAPDSTTRSA; (8) LAKGTELKPA; (9) LAIIDATKNA; (10) LAVDGAQRSA; (11) PAPQDTTKKA; (12) LPHQDTTKNA; (13) LAKDATKTIA; (14) LAKQQSASTA; (15) LAKSDQSKPA; (16) LSHQDTTKNA; (17) LAANQPSKPA; (18) LAVSDSTKAA; (19) LAAQGTAKKPA; (20) LAPDQTTRNA; (21) LAASDSTKAA; (22) LAPQDTTKNA; (23) LAKADETRPA; (24) LAHQDTAKNA; (25) LAHQDTKKNA; (26) LAHQDTTKHA; (27) LAHQDTTKKA; (28) LAHQDTTRNA; (29) LAHQDTTNA; (30) LAHQGTTKNA; (31) LAHQVTTKNA; (32) LAISDQSKPA; (33) LADATKTA; (34) LAKDTTKNA; (35) LAKSDQSRPA; (36) LAPQDTKKNA; (37) LATSDSTKAA; (38) LAVDGSQRSA; (39) LPISDQTKHA; (40) LPKDATKTIA; (41) LPPQDTTKNA; 및 (42) PAPQDTTKNA.
- 청구항 16에 있어서, 상기 펩타이드는 하기 아미노산 서열 중 하나를 포함하는, rAAV 비리온: (1) LAKDATKNA; (2) PAHQDTTKNA; (3) LAHQDTTKNA; (4) LATTSQNKPA; (5) LAISDQTKHA; (6) IARGVAPSSA; (7) LAPDSTTRSA; (8) LAKGTELKPA; (9) LAIIDATKNA; (10) LAVDGAQRSA; (11) PAPQDTTKKA; (12) LPHQDTTKNA; (13) LAKDATKTIA; (14) LAKQQSASTA; (15) LAKSDQSKPA; (16) LSHQDTTKNA; (17) LAANQPSKPA; 및 (18) LAVSDSTKAA.
- 하기를 포함하는 약제학적 조성물:
a) 청구항 1 내지 18 중 어느 한 항의 재조합 아데노-관련된 바이러스 비리온; 및
b) 약제학적으로 허용가능한 부형제. - 개체에서 유전자 산물을 망막 세포에 전달하는 방법으로서, 상기 개체에게 청구항 1 내지 18 중 어느 한 항에 따른 재조합 아데노-관련된 바이러스 (rAAV) 비리온을 투여하는 것을 포함하는, 방법.
- 청구항 20에 있어서, 상기 유전자 산물은 폴리펩타이드인, 방법.
- 청구항 20에 있어서, 상기 유전자 산물은 짧은 간섭 RNA 또는 압타머인, 방법.
- 청구항 21에 있어서, 상기 폴리펩타이드는 신경보호성 인자, 항-혈관신생 폴리펩타이드, 항-세포자멸적 인자, 또는 망막 세포의 기능을 향상시키는 폴리펩타이드인, 방법.
- 청구항 21에 있어서, 상기 폴리펩타이드는 신경교 유래된 신경친화성 인자, 섬유모세포 성장 인자 2, 뉴르투린, 섬모 신경친화성 인자, 신경 성장 인자, 뇌 유래된 신경친화성 인자, 표피 성장 인자, 로돕신, 세포자멸사의 X-연결된 억제제, 레티노쉬신, RPE65, 색소성 망막염 GTPase-상호작용 단백질-1, 페리페린, 페리페린-2, 로돕신, RdCVF, 색소성 망막염 GTPase 조절자 (RPGR), 또는 소닉 헤지혹인, 방법.
- 청구항 21에 있어서, 상기 폴리펩타이드는 RNA-유도된 엔도뉴클레아제인, 방법.
- 안구 질환을 치료하는 방법으로서 치료가 필요한 개체에게 청구항 1 내지 18 중 어느 한 항에 따른 재조합 아데노-관련된 바이러스 (rAAV) 비리온의 유효량을 투여하는 것을 포함하는, 방법.
- 청구항 26에 있어서, 상기 투여는 안구내 주사에 의한 것인, 방법.
- 청구항 26에 있어서, 상기 투여는 유리체내 주사에 의한 것인, 방법.
- 청구항 26에 있어서, 상기 안구 질환은 녹내장, 색소성 망막염, 황반 변성, 망막층간분리, 레버 선천성 흑내장, 당뇨 망막병증, 색맹, 또는 색상 시각상실인,, 방법.
- 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질을 인코딩하는 뉴클레오타이드 서열을 포함하는 단리된 핵산으로서, 상기 변이체 AAV 캡시드 단백질은 상응하는 친계 AAV 캡시드 단백질에 대해 캡시드 단백질 GH 루프에서 약 5개의 아미노산 내지 약 20개의 아미노산의 삽입을 포함하고, 그리고 상기 변이체 캡시드 단백질은, AAV 비리온 내에서 존재할 때, 망막 세포의 AAV 비리온의 증가된 감염성을 제공하고, 그리고 상기 아미노산 삽입은 천연 AAV 캡시드의 GH 루프에 있고, 상기 삽입은 식 I-X 중 임의의 것의 펩타이드인, 단리된 핵산.
- 청구항 30에 있어서, 상기 삽입 부위는 AAV2의 아미노산 587 내지 588, AAV1의 아미노산 590 내지 591, AAV5의 아미노산 575 내지 576, AAV6의 아미노산 590 내지 591, AAV7의 아미노산 589 내지 590, AAV8의 아미노산 590 내지 591, AAV9의 아미노산 588 내지 589, 또는 AAV10의 아미노산 588 내지 589인, 단리된 핵산.
- 청구항 30 또는 31의 핵산을 포함하는, 단리된, 유전자 변형된 숙주세포.
- 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질로서, 상기 변이체 AAV 캡시드 단백질은 약 5개의 아미노산 내지 약 20개의 아미노산의 삽입을 포함하고, 상기 아미노산 삽입은 천연 AAV 캡시드의 GH 루프에 있고, 상기 삽입은 식 I-X 중 임의의 것의 펩타이드인, 상기 변이체 아데노-관련된 바이러스 (AAV) 캡시드 단백질.
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