CRISPR/Cas9-based HLA immuno-engineering in human pluripotent stem cells to reduce immune rejection during cell transplantation

Abstract

환자 유래 만능 줄기 세포 (iPSCs)는 재생 의학 분야에서 응용 잠재력이 크다. 체세포 핵 치환 (SCNT) 으로부터 파생된 배아 줄기세포 (ESCs)는 수정된 배아에서 파생된 “진정한” 배아 줄기 세포를 나타내므로 iPSC 보다 많은 이점을 제공한다. 인간전분화능줄기세포 (hPSCs)로부터 분화시킨 세포의 동종 이식 성공은 환자와 기증자 세포 사이의 다형성 인간 백혈구 항원 (HLA)의 호환에 달려있다. 환자와 기증자의 HLA 유전자 사이의 복잡성 정도는 세포 치료에 대한 1차 면역 장벽을 형성한다. 따라서, CRISPR/CAS9 기술을 이용하여 기능적 항원 제시 능력을 갖춘 유전자조작 저면역원성 hPSC의 확립은 hPSC 기반 세포 치료를 크게 촉진시킬 것이다. 우리는 HLA-B 유전자의 혈청형을 변경하여 수혜자와 더 일치하는 기증자를 생성하기 위해 CRISPR/CAS9 기술을 이용한 유전자 삽입 전략을 사용하였다. 먼저, HLA-B 유전자 특이적 편집을 통해 HLA-B 제거 (HLA-B-KO) SCNT-ESC 세포주를 생성하였다. 두번째로 HLA-B KO 세포주에 수혜자와 일치하는 HLA-B 혈청형을 삽입하였다. HLA-B KO 세포주로부터 분화된 성숙 세포는 인터페론-감마 자극 후에도 세포 표면 HLA 항원을 발현하지 않는 반면, HLA-B 혈청형이 대체된 세포주는 SCNT-ESC의 다능성 특성에 영향을 미치지 않고 HLA-B 발현을 회복한 것을 관찰하였다. 이 확립된 세포주는 ‘수용자 유사’ 항원을 발현할 잠재적 능력을 가지고 있다. 이 연구를 통해 숙주 면역원성은 낮추고 이식 후 장기간에 걸친 면역억제제 치료의 필요성을 줄일 수 있다. 이 기술의 주요 이점은 CD8+ 세포에 외부 항원뿐 아니라 숙주를 제시하는 HLA-B 기능을 유지하는 것이다. 또한, 잠재적으로 세포 치료에 보편적 공여자로 사용될 수 있는 HLA 동형접합 세포주를 생성하기 위해 사용될 수 있다.|Patient-derived pluripotent stem cells (PSCs) have strong potential application in the field of regenerative medicine. Embryonic stem (ES) cells derived from somatic cell nuclear transfer (SCNT) offers advantage over iPSCs as it closely represents fertilized embryo derived “true” ES cells. The success of allogenic transplantation of hPSCS is highly influenced by the compatibility of polymorphic Human Leukocyte Antigen (HLA) between patient and donor cells. The degree of complexity between HLA gene of patient and donor forms the primary immune barrier to the cell therapy. Therefore, generation of immune-engineered hypoimmunogenic hPSCs with functional antigen presenting ability using CRISPR/Cas9 technology will greatly facilitate hPSC-based cell therapy. Herein, I demonstrated the use of CRISPR/Cas9 system in combination with gene knock-in strategies to change the serotype of HLA B gene to generate better match donors to recipients. First, I generated HLA-B knockout (HLA-B-KO) SCNT-ES cell line with HLA-B gene specific editing. Second, I knocked-in HLA-B serotype matching to recipient in HLA-B KO cell line. Differentiated mature cell from HLA-B KO cell line do not express cell surface HLA antigen even after interferon- γ stimulation, while HLA-B serotype changed cell line showed regaining of HLA-B expression without affecting pluripotency nature of the SCNTs. Moreover, the HLA-B serotype replaced stem cells were able to efficiently differentiate into lung air epithelial cells, cardiomyocytes and neural-crest like cells. This uniquely generated cell line has the potential to function as normal with ‘recipient – like’ antigen presenting properties. The host immunogenicity will be lower and can eliminate the need for immunosuppressant treatments over prolong periods following transplantations. A major advantage of this technique would be the retention of the HLA-B functionality to present host as well as pathogenic antigens to CD8+ T cells. The strategy can be extended to generate HLA- homozygous cell line that can be used as potential universal donors in stem cell therapies.|The step-wise differentiation of hematopoietic stem cells (HSCs) is required for formation of different components of blood cell types. The differentiation of human induced pluripotent stem cells (hiPSCs) to multipotent hematopoietic progenitors requires the recapitulation of appropriate development stages. The pseudogene HLA-H is associated with hereditary hemochromatosis, an autosomal recessive disorder of iron metabolism. The mutations in HLA-H gene interferes with interaction between HLA-H and beta-2-microglobulin that might results in iron overload. In this study, I investigated effect of HLA-H knockout on differentiation of hiPSCs into hematopoietic progenitor stem cells. Here, I knock-out HLA-H gene using CRISPR/Cas9 system in hiPSCs and studied its effect on hematopoietic progenitor cell differentiation. Here, I reported that CRISPR/Cas9-mediated knockout of HLA-H does not affect pluripotency characteristics of hiPSCs. Moreover, HLA-H loss significantly increased the frequency of CD34+CD45+ and CD31+CD45+ in hiPSCs derived hematopoietic progenitor cells. Furthermore, disruption of HLA-H showed increased differentiation of hiPSCs-derived CD43+CD34+ cells. Collectively these data suggest that HLA-H, might play an important role in HSC differentiation and commitment to hematopoietic lineage.