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IntroductionHemophilia A and B are the most common bleeding disorders caused by deficiencies of clotting factors VIII and IX, respectively, both of which are X-linked with a recessive heredity.1 Replacement of the deficient factors with frequent intravenous injections of plasma concentrates or recombinant proteins is the standard treatment for these diseases.2 Great efforts have been made for nearly a decade toward developing experimental gene therapy for these diseases and aiming at the development of a medical intervention that is more effective and convenient than the currently available replacement therapies.3 Hemophilia is a suitable clinical model for the development of gene therapy products and has a number of advantages: 1) there is a simple and well defined cause-and-effect relationship between the protein deficiencies and bleeding symptoms; 2) tissue-specific expression and precise regulation of the transgenes are not necessary; 3) well characterized animal models are available for preclinical studies; 4) an unequivocal endpoint for product efficacy can be assessed in clinical trials; and 5) even 1% to 5% of the normal physiological levels of the proteins is therapeutic.For gene therapy of hemophilia, the most challenging hurdle, with respect to the long-term expression of the deficient proteins at adequate levels, is the development of a suitable gene delivery system. Technologies have been evolving from ex vivo to in vivo approaches, from initial use of retroviral vector to recent application of adenviral (Ad) or adeno-associated virus (AAV) vector, demonstrating progress from early results of transient low-level expression to more sustained high-level expression.3 For hemophilia A treatment, Ad vectors are particularly useful, since the liver naturally produces factor VIII, and following intravenous (i.v.) injection, Ad vectors concentrate in the liver. This makes the gene transduction efficiency to liver very high. Adenovirus vectors have been developed for gene therapy due to their high titer, broad infectivity, potential for large payload, and in vivo gene delivery capacity.4 Although the immunogenicity and cytotoxicity associated with the early-generation Ad vectors have been a concern with respect to their clinical application, newly developed vectors, in which the viral coding sequences have been deleted, have significantly reduced the side effects associated with the vectors. The “gutless” Ad vector, or so called helper-dependent, large-capacity, or mini- Ad vectors are the representative examples of these new-generation Ad vectors.5-15 The mini-Ad vector system described in this report was developed based on two major research findings. First, an Ad- SV40 hybrid virus discovered during attempts to grow human Ad in non-permissive monkey COS-7 cells.16 The hybrid virus had a genome structure in which only both ends of the Ad sequences were retained and almost all coding sequences of the Ad genome were replaced by symmetric, tandemly repeated SV40 genomes. The hybrid viruses replicated and were packaged in the presence of a wild-type Ad as a helper. This finding implied that total replacement of the Ad genome was possible to form a mini-Ad vector as long as proper helper function and selective pressure was provided. Secondly, it was discovered that Ad packaging can be attenuated by deleting portions of the packaging signal.17 This finding provided a means to put selective pressure on the helper Ad (referred to as ancillary Ad) by specifically limiting its packaging process and allowing a preferential packaging of the mini-Ad. The system, therefore, is designed to have three main components: the mini-Ad vector, the E1-deleted ancillary Ad, and a production cell line that provides AdE1 complementation.Based on the mini-Ad vector system, MiniAdFVIII was developed. The MiniAdFVIII vector carries a 27 kb expression cassette, in which the full-length human factor VIII cDNA is flanked by a human albumin promoter and cognate genomic sequences. Infection of MiniAdFVIII in vitro showed that the vector mediated expression of functional human factor VIII at levels of 100-200 ng/106 cells per 24 hours in HepG2 and 293 cells. With single-dose intravenous injection of 1011 viral particles in hemophilic mice, MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml for up to 369 days) that corrected the factor VIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxicities in mice and dogs after a single intravenous dose of up to 3×1011 and 6×1012 viral particles, respectively. In this report, other studies for developing the MiniAdFVIII vector with a site-specific integration capability and the development of a human factor VIII-tolerized mouse model for preclinical studies of MiniAdFVIII are described.

Purpose Platelet aggregation plays a key role in dysregulation of coagulation and development of TM in pig cardiac xenografts transplanted into baboon recipients. The present study aimed to evaluate the influence of pig genetic modification on human platelet aggregation induced by pig endothelial cells (EC). Methods and Materials Using a Chrono-log Whole Blood Aggregometer, we studied platelet aggregation after incubation of human blood with human (h) or pig (p) aortic endothelial cells (AEC) using wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and various transgenic pigs expressing human complement- or coagulation-regulatory proteins. We also studied the development of TM after transplantation of hearts from two types of genetically-engineered pigs, one of which expressed hTBM. Results Platelet aggregation induced by pAEC from GTKO pigs or by pAEC expressing complement-regulatory (CD46 and/or CD55) and/or coagulation-regulatory (TFPI or TBM) proteins was significantly reduced, particularly by GTKO.CD46.TBM pAEC (WT 53% vs GTKO.CD46.TBM 28%; p 3 /mm 3 or no reduction in platelet count or fibrinogen was observed. Two experiments are ongoing, but one baboon was euthanized after 52 days; there was no TM on histology. Conclusions The absence of the Gal antigen, and transgenic expression of human complement- and/or coagulation-regulatory proteins on pAEC are all associated with reduced human platelet aggregation in vitro. Transplantation of hearts expressing TBM was associated with no (or possibly delayed) TM. The barrier of coagulation dysregulation may be overcome.

Purpose Analyses of xenogenic GalTKO.hCD46 organ perfusions have revealed that activation of complement cascade and platelets play a substantial role in rejection of the perfused organ. With the aim of better controlling these mechanisms, human endothelial protein C receptor (hEPCR), a receptor facilitating protein C activation by the thrombin-thrombomodulin complex, has been added on the genetic GalTKO.hCD46 background. Here we evaluate effects of this additional genetic modification in a xenogenic lung perfusion model. Methods and Materials 6 GalTKO.hCD46.hEPCR and 37 GalTKO.hCD46 pig lungs were perfused with heparinized fresh human blood until failure (by oxygenation or PA flow and pressure criteria) or elective termination at 4 h. Functional parameters (oxygenation, PVR) were recorded, and blood samples were collected at pre-specified intervals throughout the perfusion for analysis of cell counts and cell activation. Results Median survival time in GalTKO.hCD46 organs was 171’ whereas GalTKO.hCD46.hEPCR lungs all “survived” to an elective termination (MST 240’, p=0.006). Platelet activation was significantly lower in the GalTKO.hCD46.hEPCR group (ΔBTG at 2h: 119±163 vs. 770±377, p=0.008). However, platelet and neutrophil sequestration was not prevented by the additional hEPCR expression. Thrombin generation was lower throughout the perfusion in hEPCR lungs without reaching statistical significance. PVR was lowered in hEPCR organs when compared to the GalTKO.hCD46 controls (PVR at 90’: 76±81 vs. 171±110, p=0.0536). Conclusions Results of this study demonstrate that genetic modifications to the pig, targeting anticoagulant mechanisms, do suppress platelet activation and lower activation of the complement cascade. As a consequence, PVR was lowered and survival was significantly improved. Expression of hEPCR, and perhaps other anticoagulant molecules, may facilitate successful lung xenotransplantation.

Background: Islets obtained from genetically-engineered (GE) pigs with Gal-knockout (GTKO) and high expression of hCD46 have shown long-term function and successful correction of insulin independence in nonhuman primates (NHP). Two anti-coagulant genes (hTFPI and hCD39), and an immunosuppressive gene (pCTLA4Ig) have been added to the GTKO/hCD46 background, using an islet-specific expression system, for inhibition of thrombosis, inflammation, early islet loss, and rejection. Transplant outcomes are being tested in a diabetic monkey model using adult islets from these multi-transgenic pigs. Methods: Three individual vectors (CD39ins, TFPIins and CTLA4Igins), each under control of the rat insulin II promoter and mouse PDX-1 enhancer, were transfected alone or in combination into GTKO/hCD46 pig fibroblast cells. GTKO/hCD46/CD39ins, GTKO/hCD46/TFPIins/CTLA4Igins, and GTKO/hCD46/CD39ins/TFPIins/CTLA4Igins cell lines were used for nuclear transfer (NT) to produce pregnancies. Cloned pigs were analyzed for transgene expression in pancreas, heart, and liver by IHC, Western, and real-time PCR. Glucose metabolism in the various GE pigs was determined by measurement of blood glucose, c-peptide, and via intravenous glucose tolerance tests (ivGTT) or arginine stimulation test. GE islets from adult pigs were transplanted intraportally into STZ-diabetic monkeys using an ATG/MMF/anti-CD154 immunosuppression regimen. Results: Viable multi-transgenic pigs with three, four, or five different genetic modifications were produced. Three different pig lines were established: (i) GTKO/hCD46/CD39ins, (ii) GTKO/hCD46/TFPIins/CTLA4Igins, and (iii) GTKO/hCD46/CD39ins/TFPIins/CTLA4Igins. IHC revealed robust islet-specific expression of CD39, TFPI and CTLA4Ig in pancreas, with only background expression in heart and liver. Western analysis showed strong pCTLA4Ig expression only in the pancreas. Blood glucose metabolism was normal in all GE pigs tested. Islets from GE pigs containing the TFPI transgene, when mixed with human blood, showed prolonged clotting times. Early islet loss (IBMIR) was reduced 5–7 fold in NHP transplants using islets from 4-GE and 5-GE pigs vs. 2-GE pig islets. Conclusions: (i) Healthy multi-transgenic pigs with islet-specific expression of hCD39, hTFPI, and pCTLA4Ig were produced. (ii) Glucose metabolism was normal in these GE pigs. (iii) GE islets showed anti-coagulation activity in vitro, and protection from IBMIR in vivo in NHP transplants. (iv) GE islets transplanted into diabetic monkeys demonstrated prolonged survival, function, and complete normalization of blood glucose levels for up to 1 year.

Successful development of somatic cell nuclear transfer (cloning) technology in pigs has allowed for precise genetic manipulation of the pig genome. For xenotransplantation applications, pigs have been produced in which both copies of the α1,3-galactosyl transferase (GT) gene were inactivated (GTKO pigs). Analysis of tissues from GTKO pigs demonstrated a complete lack of immunogenic Galα1,3Gal (Gal) sugars, while in vivo pre-clinical studies in nonhuman primates, using cells (i.e. pancreatic islets) or whole organs (heart, kidney, liver, lung), demonstrated the elimination of hyperacute rejection, and prolonged survival compared to wild-type controls. While survival of GTKO xenografts was extended, challenges including induced antibody responses to non-gal antigens, thrombosis, inflammation, and cell-mediated rejection remained, pointing to the need for further genetic modification of the source pig. Towards this goal, through a combination of cloning and breeding, in combination with GTKO, we have produced multi-transgenic pigs (some with 5 different transgenes) with controlled expression of genes for (1) complement regulation to address the humoral response to anti-non-gal targets (DAF, CD46); (2) inhibition of inflammation and thrombosis (TFPI, CD39, thrombomodulin, EPCR); and (3) local protection against the human cellular response (CTLA4Ig, CIITA-DN). For some transgenes a constitutive promoter system can be used for expression in all tissues, such that one animal can be used for multiple transplant applications, however, our results have shown that for certain transgenes, tissue-specific gene expression is preferred. Since inhibition of thrombosis, complement modulation, and suppression of T-cell responses are important to delayed xenograft rejection of both whole organs and islet cell xenografts, pigs have been produced with tissue-specific transgene expression in either the vascular endothelium or endocrine pancreas compartments, or constitutively in all tissues. In vivo results in nonhuman primates have demonstrated complete normalization of blood glucose for up to 1 year in diabetic monkeys, and 8-month survival of multigenic pig hearts in baboons, evidence for the promise of this technology for human clinical applications.

David KC Cooper,1 David Ayares21Thomas E Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 2Revivicor, Blacksburg, VA, USAAbstract: The transplantation of organs and cells from pigs into humans could overcome the critical and continuing problem of the lack of availability of deceased human organs and cells for clinical transplantation. Developments in the genetic engineering of pigs have enabled considerable progress to be made in the experimental laboratory in overcoming the immune barriers to successful xenotransplantation. With regard to pig organ xenotransplantation, antibody- and cell-mediated rejection have largely been overcome, and the current major barrier is the development of coagulation dysregulation. This is believed to be due to a combination of immune activation of the vascular endothelial cells of the graft and molecular incompatibilities between the pig and primate coagulation–anticoagulation systems. Pigs with new genetic modifications specifically directed to this problem are now becoming available. With regard to less complex tissues, such as islets (for the treatment of diabetes), neuronal cells (for the treatment of Parkinson's disease), and corneas, the remaining barriers are less problematic, and graft survival in nonhuman primate models extends for >1 year in all three cases. In planning the initial clinical trials, consideration will be concentrated on the risk–benefit ratio, based to a large extent on the results of preclinical studies in nonhuman primates. If the benefit to the patient is anticipated to be high, eg, insulin-independent control of glycemia, and the potential risks low, eg, minimal risk of transfer of a porcine infectious agent, then a clinical trial would be justified.Keywords: infection, pigs, genetically-engineered, xenotransplantation, islets, xenotransplantation, organs

IntroductionHyperacute (HAR) and delayed xenograft (DXR) rejection are well defined barriers following discordant transplantation of porcine organs into primate recipients. Significant progress has been made in recent years to overcome these immunological barriers, based on the development of pigs genetically modified to reduce immunogenicity: [1,2] A major step has been the generation of pigs lacking expression of Gala1,3Gal (GalTKO), the principal carbohydrate target of human anti-pig antibody. [3,4] However, both in vitro and in vivo studies demonstrated that non-gal antibodies have the ability to activate endothelial cells, and thus, amplify procoagulant molecular incompatibilities between pig and primate.[5,6] Based on our prior work with pigs expressing the Gal epitope, we tested the hypothesis that over-expression of human complement regulatory proteins (hCRP) and/or blockade of the CD154/CD40 costimulation pathway will result in reduced graft injury, anti-pig responses and coagulation dysregulation. Briefly, twenty two organs (13 hearts, 9 kidneys) from GalT-KO pigs (9 expressing hCRP) were transplanted into baboons that received no immunosuppressive therapy, or a therapeutic regimen based on costimulation blockade. The incidence of HAR was decreased in GalTKO organs expressing hCRP as compared to GalTKO. Pre-transplant anti-non-Gal antibody titers were inconsistently associated with early GalT-KO xenograft failure. In contrast, coagulation cascade activation and platelet activation correlated closely with incidence of HAR of GalTKO organs. Strong elicited anti-non-gal antibody responses were detected after transplantation of a GalTKO organ. Delayed xenograft rejection was associated with coagulation dysregulation and elicited anti-non-gal antibody production. Future work in xenotransplantation should place emphasis on further targeting of anti-non gal humoral immune responses and inhibiting coagulation activation. References 1. Pierson RN III. JAMA 2009; Current status of xenotransplantation. 30: 967–969. 2. Cooper DK, Dorling A, Pierson RN III, et al. Alpha1,3-galactosyltransferase gene-knockout pigs for xenotransplantation: where do we go from here? Transplantation 2007; 84: 1–7. 3. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 2005; 11: 32–34. 4. Kuwaki K, Tseng YL, Dor FJ, et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med 2005; 11: 29–31. 5 Saethre M, Baumann BC, Fung M, Seebach JD, Mollnes TE. Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells. Transplantation. 2007; 84: 244–250. 6. Hisashi Y, Yamada K, Kuwaki K, et al. Rejection of cardiac xenografts transplanted from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs to baboons. Am J Transplant. 2008; 8: 2516–2526.

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as of cloned embryos. The objective of this study was to identify and sequence isoforms of Dnmt1 expressed in porcine oocytes. Total RNA was isolated from pools of 50 denuded mature oocytes as well as from fibroblast cells using the Trizol method. RNA was co-precipitated with glycogen, and residual genomic DNA was removed with DNase I. A RACE System (Invitrogen, Carlsbad, CA, USA) was used to amplify the 5′ cDNA end of Dnmt1. Briefly, the first strand of cDNA was synthesized using SuperScript II and a Dnmt1-specific primer followed by degradation of the RNA strands and incorporation of TdT and dCTP tails to the 3′ ends of the cDNA. Tailed cDNA was amplified by PCR using a forward anchor primer and a reverse Dnmt1-specific primer. PCR products were separated by electrophoresis on an agarose gel. Resulting PCR products were subcloned into a cloning vector, and the cDNA inserts were sequenced. PCR primers capable of amplifying all possible alternatively spliced isoforms of Dnmt1 were used to identify the presence of the RNA sequences found in fibroblasts and oocytes of pigs. Two distinctive bands (290 and 390 bp) were observed after 5′RACE, PCR, and electrophoresis of oocyte Dnmt1 cDNA. Only 1 band of 290 bp was observed after amplification of fibroblast cDNA. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa Dnmt1 genomic sequence located on chromosome 2 (CU462940). The smaller 290-bp band, amplified from both oocytes and fibroblasts, had an identical DNA sequence (EU908731). Structure analysis of the larger 390-bp band indicates that this oocyte Dnmt1 isoform has an additional exon located between exon 1 and 2 of the somatic form of Dnmt1 (EU908730). PCR products amplified using primers specific for the oocyte or somatic transcript verified the presence of the additional exon in the oocyte Dnmt1 splicing variant. In conclusion, this study shows that porcine oocytes express an alternative isoform of Dnmt1 in addition to the somatic transcript. The 2 identified isoforms are produced by alternative splicing of the Dnmt1 gene. Analysis of the oocyte and somatic Dnmt1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

Different from cloned mice and cattle, which are often born with large offspring syndrome (LOS), pigs produced through nuclear transfer have not been found to be overly large compared to their age-matched controls. In our study, significant differences (P < 0.05) were found in the body weight of both deceased newborn cloned piglets (n = 9) (1.2 � 0.8 vs. 2.1 � 0.2 kg) and cloned pigs at one month of age (n = 7) (6.1 � 1.3 vs. 8.0 � 0.8 kg), when compared to their controls (n = 5 for each age group). Because imprinted genes are important regulators of fetal growth and are subject to reprogramming during nuclear transfer, we aimed to determine the expression levels of both growth-enhancing and growth-inhibiting imprinted genes in these cloned pigs by real time quantitative reverse transcription polymerase chain reaction (RT-PCR). These genes include: Igf2 and Peg3 (growth-promoting), and Igf2r and Grb10 (growth-inhibiting). Tissues from six major organs were collected from clones and controls of both age groups. SYBR Green I was utilized as a reporter for the PCR reactions and 18S ribosomal RNA was used as the internal reference for quantifying the relative expression level of all genes. With the exception of Igf2, the other three imprinted genes were found to have statistically different levels of expression in certain organs of both groups of pigs when compared to their age-matched controls. However, no strong correlation was found between the levels of gene expression and the low-body-weight phenotype of the cloned pigs. Contrary to the notion that nuclear transfer can reduce the variation in the resulting animals due to their identical genetic make-up, larger variances of gene expression were found in clones of both groups, indicating the randomness of the nuclear reprogramming process. For all four imprinted genes studied, the coefficient of variation (CV) decreased greatly from newborn to one month old in both clones and controls, suggesting a more stringent regulation on imprinted gene expression after birth.

We have examined the homology requirements for intermolecular recombination between plasmids introduced into human, monkey, and bacterial cells. Variable-size-deletion derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo were constructed. Each of these plasmids was mixed with another pSV2neo plasmid containing a different, nonoverlapping deletion. Recombination was measured in mammalian cells and bacteria by the frequency of reconstruction of an intact neo gene. We observed that 25 base pairs of homologous sequence is sufficient to yield recombinant products, implying that synapsis and homologous pairing can occur with this level of homology. Examination of the products revealed that nonreciprocal recombination played a role in the generation of normal neo genes. In addition coconversion of linked markers was observed. Exonucleolytic action seems to play a role in gene conversion.

We studied the ability of mammalian cells to repair single-stranded nicks, gaps, and loops in DNA duplexes. Heteroduplexes prepared from derivatives of the shuttle vector pSV2neo were introduced into monkey COS cells. After replication, the plasmids were recovered and used to transform Escherichia coli. Plasmid DNA from the recovered colonies was tested for repair at each of six different sites. We observed that mammalian cells are capable of repairing single-stranded gaps and free single-stranded ends most efficiently. Regions containing twin loops were recognized, and one of the loops was excised. Portions of the molecules containing small single loops were also repaired. Markers which were 58 nucleotides apart were corepaired with nearly 100% efficiency, while markers which were 1,000 nucleotides or more apart were never corepaired. The mechanisms involved in heteroduplex repair in mammalian cells seem to be similar to those involved in repairing DNA lesions caused by physical and chemical agents.

We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.

Objectives Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. Methods Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents. Results Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. Conclusions Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.

The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.

Uniformly methylated heteroduplex plasmids which contained 6 mismatched regions, including loops of 24, 30, 248 and 283 nucleotides, as well as single-stranded gaps and free ends were introduced into a recombination-deficient strain of bacteria, and the products of repair were analyzed. The results indicate that these cells are capable of repairing all of these structures, although with different efficiencies. Repair of single-stranded gaps and free ends, which occurs most efficiently, is always associated with acquisition of information from the uncut strand (unidirectional repair). Regions containing single loops or twin loops were repaired at similar efficiencies. In these cases each of the two strands was capable of acting as the template for repair (bidirectional repair). At sites containing twin or substitution loops, the larger of the loops was removed twice as efficiently as the smaller loop. DNA sequencing of the repaired regions indicated that the repair is precise. The data also suggest that markers separated by only 58 nucleotides do not always segregate together indicating that repair tracts may be relatively short.

If an ABO-incompatible heart is transplanted into an infant before natural antibodies have developed to the specific donor carbohydrate A/B antigen(s), then B-cell tolerance to the donor A/B antigen is achieved, and these antibodies never develop. Anti-carbohydrate antibodies play a role in the rejection of wild type (WT) and alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig xenografts. We investigated development of these antibodies in infant baboons and humans. Serum samples from infant baboons (n = 42) and humans (n = 42) were tested by flow cytometry for immunoglobulin M and immunoglobulin G binding to peripheral blood mononuclear cells from WT and GT-KO pigs, and for complement-dependent cytotoxicity. The presence of anti-blood group antibodies was tested in baboon serum. In infant baboons and humans, cytotoxic anti-Galalpha1,3Gal antibodies develop during the first 3 months, and steadily increase with age, whereas cytotoxic anti-nonGal antibodies are either absent or minimal in the majority of cases throughout the first year of life. Anti-blood group antibodies were not detected before 16 weeks of age. Our data suggest GT-KO pig organ/cell transplants could be carried out in early infancy in the absence of preformed cytotoxic anti-nonGalalpha1,3Gal antibodies.