lentiviral-vector-mediated-gene-therapy-hepatocytes-ex-vivo-for

Lentiviral Vector-mediated Gene Therapy of Hepatocytes <em>Ex Vivo</em> for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Mayo Clinic

In this investigation we studied the efficacy and durability of recombinant adeno-associated virus serotype 9 (rAAV9) vector-mediated gene transfer to the transplanted rat heart.

Efficient and durable gene transfer to transplanted heart using adeno-associated virus 9 vector.

&Alpha;1, 3-galattosiltransferasi gene knockout (GTKO) maiali ridotto il significato di anticorpi agli antigeni di galattosio alfa 1,3-galattosio (Gal) ma non eliminavano reiezione xenoinnesto ritardato (DXR). Ipotizziamo che risultati organi DXR di GTKO da una risposta anticorpale a un numero limitato di non-Gal endothelial cell gli antigeni di membrana (CE). In questo studio, abbiamo proiettato una libreria di espressione retrovirus per identificare gli antigeni di membrana CE rilevati dopo lo xenotrapianto cardiaco.

Identificazione di nuovi antigeni proteine carboidrati e membrana in xenotrapianto cardiaco.

&Alpha;1&#xFF0C;3 &#x8F6C;&#x79FB;&#x57FA;&#x56E0;&#x6572;&#x9664; &#xFF08;GTKO&#xFF09; &#x732A;&#x51CF;&#x5C11;&#x534A;&#x4E73;&#x7CD6; alpha 1&#xFF0C;3-&#x534A;&#x4E73;&#x7CD6; (Gal) &#x6297;&#x539F;&#x6297;&#x4F53;&#x7684;&#x610F;&#x4E49;&#xFF0C;&#x4F46;&#x6CA1;&#x6709;&#x6D88;&#x9664;&#x5EF6;&#x8FDF;&#x7684;&#x5F02;&#x79CD; (DXR)&#x3002;&#x6211;&#x4EEC;&#x63A8;&#x6D4B; GTKO DXR &#x673A;&#x5173;&#x7ED3;&#x679C;&#x4ECE;&#x6297;&#x4F53;&#x53CD;&#x5E94;&#x5230;&#x4E3A;&#x6570;&#x6709;&#x9650;&#x7684;&#x975E; Gal &#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE; (EC) &#x819C;&#x6297;&#x539F;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x6211;&#x4EEC;&#x7504;&#x522B;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x8868;&#x8FBE;&#x5E93;&#xFF0C;&#x4EE5;&#x8BC6;&#x522B;&#x5FC3;&#x810F;&#x5F02;&#x79CD;&#x79FB;&#x690D;&#x540E;&#x68C0;&#x6D4B;&#x5230;&#x7684; EC &#x819C;&#x6297;&#x539F;&#x3002;

&#x5FC3;&#x810F;&#x5F02;&#x79CD;&#x79FB;&#x690D;&#x65B0;&#x78B3;&#x6C34;&#x5316;&#x5408;&#x7269;&#x548C;&#x819C;&#x86CB;&#x767D;&#x6297;&#x539F;&#x7684;&#x9274;&#x5B9A;&#x3002;

Î±1,3-Galactosyltransferase gene knockout (GTKO) pigs reduced the significance of antibody to galactose alpha 1,3-galactose (Gal) antigens but did not eliminate delayed xenograft rejection (DXR). We hypothesize that DXR of GTKO organs results from an antibody response to a limited number of non-Gal endothelial cell (EC) membrane antigens. In this study, we screened a retrovirus expression library to identify EC membrane antigens detected after cardiac xenotransplantation.

Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation.

&Alpha;1, 3-galactosyltransf&eacute;rase porcs de g&egrave;ne knockout (GTKO) r&eacute;duit l&#39;importance des anticorps aux antig&egrave;nes de galactose alpha 1,3-galactose (Gal) mais n&#39;&eacute;limine pas de rejet retard&eacute; x&eacute;nogreffe (DXR). Nous &eacute;mettons l&#39;hypoth&egrave;se que DXR de GTKO organes d&eacute;coule une r&eacute;action immunitaire &agrave; un nombre limit&eacute; de non-Gal endothelial cell les antig&egrave;nes membranaires (EC). Dans cette &eacute;tude, nous avons projet&eacute; une biblioth&egrave;que d&#39;expression r&eacute;trovirus afin d&#39;identifier les antig&egrave;nes membranaires EC d&eacute;tect&eacute;es apr&egrave;s la x&eacute;notransplantation cardiaque.

Identification de nouveaux antig&egrave;nes de prot&eacute;ines glucides et membrane dans la x&eacute;notransplantation cardiaque.

&Alpha;1, 3-Galaktosyltransferase gen ko (GTKO) Schweine reduziert die Bedeutung von Antik&ouml;rpern gegen Galaktose alpha 1,3-Galaktose (Gal) Antigene aber nicht beseitigen, verz&ouml;gerte Xenograft-Ablehnung (DXR). Wir vermuten, dass DXR der GTKO Organe Ergebnisse aus einer Antik&ouml;rper-Reaktion auf eine begrenzte Anzahl von nicht-Gal endothelial Membran-Antigene (EG Zelle). In dieser Studie sehen wir eine Retroviren-Ausdruck-Bibliothek zur EG-Membran-Antigene erkannt nach kardialen Xenotransplantation zu identifizieren.

Identifizierung von neuen Kohlenhydrat und Membran-Protein-Antigene in der kardialen Xenotransplantation.

&Alpha; 1, 3-&#x30AC;&#x30E9;&#x30AF;&#x30C8;&#x30FC;&#x30B9;&#x8EE2;&#x79FB;&#x9175;&#x7D20;&#x907A;&#x4F1D;&#x5B50;&#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#xFF08;GTKO&#xFF09; &#x8C5A;&#x30AC;&#x30E9;&#x30AF;&#x30C8;&#x30FC;&#x30B9; &#x30A2;&#x30EB;&#x30D5;&#x30A1; 1, 3-&#x30AC;&#x30E9;&#x30AF;&#x30C8;&#x30FC;&#x30B9; (Gal) &#x6297;&#x539F;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x6297;&#x4F53;&#x306E;&#x610F;&#x7FA9;&#x3092;&#x6E1B;&#x5C11;&#x304C;&#x9045;&#x5EF6;&#x7570;&#x7A2E;&#x79FB;&#x690D;&#x62D2;&#x7D76;&#x53CD;&#x5FDC; (DXR) &#x3092;&#x6392;&#x9664;&#x3057;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;&#x7D30;&#x80DE; (EC) &#x819C;&#x306E;&#x6297;&#x539F;&#x3092; GTKO DXR &#x81D3;&#x5668;&#x6210;&#x7E3E;&#x304B;&#x3089;&#x6297;&#x4F53;&#x5FDC;&#x7B54;&#x975E; Gal &#x5185;&#x76AE;&#x306E;&#x9650;&#x3089;&#x308C;&#x305F;&#x6570;&#x3092;&#x4EEE;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x5FC3;&#x81D3;&#x7570;&#x7A2E;&#x79FB;&#x690D;&#x5F8C;&#x691C;&#x51FA;&#x3055;&#x308C;&#x308B; EC &#x819C;&#x6297;&#x539F;&#x306E;&#x8B58;&#x5225;&#x306B;&#x306F;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x8868;&#x73FE;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x3092;&#x9078;&#x5225;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x5FC3;&#x81D3;&#x306E;&#x7570;&#x7A2E;&#x79FB;&#x690D;&#x306E;&#x65B0;&#x3057;&#x3044;&#x70AD;&#x6C34;&#x5316;&#x7269;&#x304A;&#x3088;&#x3073;&#x819C;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x6297;&#x539F;&#x306E;&#x540C;&#x5B9A;

&Alpha;1, 3-Galactosyltransferase &#xC720;&#xC804;&#xC790; &#xB179;&#xC544;&#xC6C3; (GTKO) &#xD53C;&#xADF8; galactose &#xC54C;&#xD30C; 1, 3-galactose (Gal) &#xD56D; &#xC6D0;&#xC5D0; &#xD56D; &#xCCB4;&#xC758; &#xC911;&#xC694;&#xC131;&#xC744; &#xAC10;&#xC18C; &#xD558;&#xC9C0;&#xB9CC; &#xC9C0;&#xC5F0;&#xB41C; &#xB1CC;&#xC885;&#xC591; &#xC81C;&#xAC70; (DXR)&#xB97C; &#xC81C;&#xAC70; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC81C;&#xD55C; &#xB41C; &#xBE44; Gal &#xB0B4; &#xD53C;&#xC5D0; &#xD56D; &#xCCB4; &#xC751;&#xB2F5; DXR GTKO &#xC7A5;&#xAE30; &#xACB0;&#xACFC; (EC) &#xB9C9; &#xD56D; &#xC6D0; &#xC138;&#xD3EC; &#xAC00;&#xC124;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xC2EC;&#xC7A5; xenotransplantation &#xD6C4; &#xAC10;&#xC9C0; EC &#xB9C9; &#xD56D; &#xC6D0;&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xB808;&#xD2B8; &#xC2DD; &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC; &#xC0C1;&#xC601;.

&#xC2EC;&#xC7A5; xenotransplantation&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xB9C9;&#xACFC; &#xD0C4;&#xC218;&#xD654;&#xBB3C; &#xB2E8;&#xBC31;&#xC9C8; &#xD56D;&#xC758; &#xC2DD;&#xBCC4;.

&Alpha;1, cerdos de octavos de final (GTKO) 3-Galactosyltransferase gene redujo la importancia de los anticuerpos a los ant&iacute;genos de galactosa alfa 1, 3-galactosa (Gal), pero no elimin&oacute; rechazo retardado xenoinjerto (DXR). Presumimos que DXR de GTKO resultados de &oacute;rganos de una respuesta de anticuerpos a un n&uacute;mero limitado de no-Gal endotelial celular ant&iacute;genos de membrana (CE). En este estudio, defendimos una biblioteca de expresi&oacute;n retrovirus para identificar ant&iacute;genos de membrana CE detectados despu&eacute;s de xenotrasplante card&iacute;aco.

Identificaci&oacute;n de nuevos ant&iacute;genos de la prote&iacute;na del carbohidrato y de membrana en xenotrasplante card&iacute;aco.

Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation.

Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts.

Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance.

Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.

Rejection of Gal-free (GTKO) donor pig cardiac xenografts is strongly associated with vascular non-Gal antibody binding, endothelial cell (EC) injury, and activation and microvascular thrombosis. We adopted a pig-to-SCID/beige small animal transplant model to compare the pathogenicity of baboon and human anti-pig antibody.

A pig-to-mouse coronary artery transplantation model for investigating the pathogenicity of anti-pig antibody.

Analysis of non-Gal antibody induced after pig-to-baboon cardiac xenotransplantation identified the glycan produced by porcine beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti-SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti-SDa antibody and increased binding of Dolichos biflorus agglutinin (DBA), a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA, suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2-KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha-1,3-galactosyltransferase deficient (GGTA1-KO) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient (CMAH-KO), show reduced antibody binding, confirming the presence of B4GALNT2-dependent antibodies in both humans and non-human primates. Preclinical xenotransplantation using B4GALNT2-deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti-pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.

B4GALNT2 and xenotransplantation: A newly appreciated xenogeneic antigen.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1.

Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single-cell or spheroid hepatocytes.

Article	Year
Efficient and durable gene transfer to transplanted heart using adeno-associated virus 9 vector. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation\| PubMed ID: 18442723	2008
Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation. Transplantation\| PubMed ID: 21119562	2011
Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts. Transplantation\| PubMed ID: 22391577	2012
Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation\| PubMed ID: 25176027	2014
A pig-to-mouse coronary artery transplantation model for investigating the pathogenicity of anti-pig antibody. Xenotransplantation\| PubMed ID: 26490445	2015
B4GALNT2 and xenotransplantation: A newly appreciated xenogeneic antigen. Xenotransplantation\| PubMed ID: 29604134	2018
Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1. Human gene therapy\| PubMed ID: 29764210	2018
Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models. Surgery\| PubMed ID: 29884476	2018

Zeji Du

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