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Correction of murine models of β-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human γ-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with β-thalassemic HSCs transduced with a γ-globin/MGMT vector initially had subtherapeutic levels of red cells expressing γ-globin. To enrich γ-globin–expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of γ-globin–expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of γ-globin–expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for β-thalassemia.

Correction of murine models of β-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human γ-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with β-thalassemic HSCs transduced with a γ-globin/MGMT vector initially had subtherapeutic levels of red cells expressing γ-globin. To enrich γ-globin–expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of γ-globin–expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of γ-globin–expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for β-thalassemia.

Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5′-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N -(5′-phosphopyridoxyl)- l -lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4′-Nα double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent α,β-elimination reaction of β-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic d -amino acids and oxo acids ( k cat ′=0.42 min with d -alanine at 25 °C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at Cα to be broken lies together with the Cα&z.sbnd;N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The α-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10 times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k cat (Ab·PLP) ′/ k cat (PLP) ′=5×10). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.

The pyridoxal-5'-phosphate (vitamin B6)-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B6 enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B6 enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the α family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller β family (tryptophan synthase β as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B6 enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B6 enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:436–447, 2001

The pyridoxal-5'-phosphate (vitamin B6)-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B6enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B6enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the a family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller ß family (tryptophan synthase ß as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B6enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B6enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:436–447, 2001

The pyridoxal-5′-phosphate-dependent enzymes (B6 enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B6 enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B6 enzymes with different folds, i.e., the large α family, the β family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the α family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the α family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B6 enzymes.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146–159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence‐based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F‐Zscore of the target family, expressed in “effective standard deviations” of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6‐dependent enzymes, (β/α)8‐barrel proteins, β‐trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures. Proteins 1999;35:387–400. © 1999 Wiley‐Liss, Inc.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146–159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in “effective standard deviations” of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (β/α)8-barrel proteins, β-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures. Proteins 1999;35:387–400. © 1999 Wiley-Liss, Inc.

No relevant conflicts of interest to declare.

Abstract 670This icon denotes a clinically relevant abstract

Recent genome-wide analyses of acute lymphoblastic leukemia (ALL) have identified genetic alterations targeting the B lymphoid transcription factor PAX5 in over 30% of B-progenitor ALL (Nature2007;446:758; Nature2008;453:110). The PAX5 mutations include deletions, focal internal amplification, sequence mutations and translocations that result in PAX5 haploinsufficiency or generate PAX5 mutants with impaired DNA-binding or transactivating activitiy. In almost all B-ALL cases, the mutations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor in B-ALL. To test this hypothesis, we have performed mutagenesis screens of mice heterozygous for a Pax5 null allele (Pax5+/−; Cell1994;79:901). In a chemical mutagenesis screen, thymectomized C57BL6/Sv129 Pax5+/− (N=25) and wild type (Pax5+/+; N=20) mice received a single 100mg/kg dose of the alkylating agent N-ethyl-S-nitrosourea (ENU) at 4–5 weeks of age. Pax5+/− animals exhibited a markedly increased frequency of leukemia in comparison to wild type animals. After one year, 24 of 25 Pax5+/−animals developed leukemia (median latency 246 days), compared to 3 of 20 Pax5+/+ animals (P<0.0001, Fig. 1). Furthermore, 20 of 22 evaluable Pax5+/− tumors were of B-progenitor cell lineage, in contrast to the Pax5+/+ tumors which were either myeloid (N=2) or T-lymphoid (N=1). Moreover, the Pax5+/−tumors exhibited a range of maturation from pro-B (B220+Cd19-) to pre-B (B220+Cd19+), suggesting the acquisition of additional lesions targeting the B-cell developmental pathway in the less mature tumors. In a complementary retroviral mutagenesis study, thymectomized Pax5+/−(N=32) and Pax5+/+ (N=47) mice received a single dose of Moloney murine leukemia virus at 1–2 days after birth. Pax5+/− mice displayed a higher penetrance and reduced latency of leukemia than Pax5+/+ mice (97% of Pax5+/− mice developed leukemia with median latency of 192 days, v. 66% of Pax5+/+ mice, latency 299 days, P<0.0001). Again, a significantly higher proportion of Pax5+/− tumors (23 of 29 evaluable) were B-progenitor cell lineage than the Pax5+/+ tumors (12 of 26; P=0.01; Fig. 2), and the B-lineage tumors exhibited a spectrum of maturation from pro- to pre-B cell. Cloning and capillary sequencing of retroviral integration sites identified recurring insertions targeting the DNA polymerase gene Poln, the tumor suppressor Wwox, the tyrosine phosphatase Ptprc, the cyclin gene Ccnd3, and orthologs of genes targeted by DNA copy number alterations in human B-ALL including Tbl1xr1. Together, these data indicate that a decrease in the normal transcriptional level of Pax5 directly contributes to the development of B-progenitor ALL, consistent with Pax5 functioning as a haploinsufficient tumor suppressor. Next-generation sequencing to comprehensively identify all genes targeted by retroviral insertions, coupled with genome-wide copy number analysis, are in progress and should provide invaluable information on the range of mutations that cooperate with Pax5 haploinsufficincy to induce ALL. Fig. 1 ENU mutagenesis Fig. 1. ENU mutagenesis Fig. 2 B-cell tumors in MoMuLV infected mice Fig. 2. B-cell tumors in MoMuLV infected mice

Recent genome-wide analyses of acute lymphoblastic leukemia (ALL) have identified genetic alterations targeting the B lymphoid transcription factor PAX5in over 30% of B-progenitor ALL (Nature 2007; 446:758; Nature 2008; 453:110). The PAX5mutations include deletions, focal internal amplification, sequence mutations and translocations that result in PAX5haploinsufficiency or generate PAX5mutants with impaired DNA-binding or transactivating activitiy. In almost all B-ALL cases, the mutations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor in B-ALL. To test this hypothesis, we have performed mutagenesis screens of mice heterozygous for a Pax5null allele (Pax5+/−; Cell 1994; 79:901). In a chemical mutagenesis screen, thymectomized C57BL6/Sv129 Pax5+/−(N=25) and wild type (Pax5+/+; N=20) mice received a single 100mg/kg dose of the alkylating agent N-ethyl-S-nitrosourea (ENU) at 4–5 weeks of age. Pax5+/−animals exhibited a markedly increased frequency of leukemia in comparison to wild type animals. After one year, 24 of 25 Pax5+/−animals developed leukemia (median latency 246 days), compared to 3 of 20 Pax5+/+animals (P<0.0001, Fig. 1). Furthermore, 20 of 22 evaluable Pax5+/−tumors were of B-progenitor cell lineage, in contrast to the Pax5+/+tumors which were either myeloid (N=2) or T-lymphoid (N=1). Moreover, the Pax5+/−tumors exhibited a range of maturation from pro-B (B220+Cd19-) to pre-B (B220+Cd19+), suggesting the acquisition of additional lesions targeting the B-cell developmental pathway in the less mature tumors. In a complementary retroviral mutagenesis study, thymectomized Pax5+/−(N=32) and Pax5+/+(N=47) mice received a single dose of Moloney murine leukemia virus at 1–2 days after birth. Pax5+/−mice displayed a higher penetrance and reduced latency of leukemia than Pax5+/+mice (97% of Pax5+/−mice developed leukemia with median latency of 192 days, v. 66% of Pax5+/+mice, latency 299 days, P<0.0001). Again, a significantly higher proportion of Pax5+/−tumors (23 of 29 evaluable) were B-progenitor cell lineage than the Pax5+/+tumors (12 of 26; P=0.01; Fig. 2), and the B-lineage tumors exhibited a spectrum of maturation from pro- to pre-B cell. Cloning and capillary sequencing of retroviral integration sites identified recurring insertions targeting the DNA polymerase gene Poln, the tumor suppressor Wwox, the tyrosine phosphatase Ptprc, the cyclin gene Ccnd3, and orthologs of genes targeted by DNA copy number alterations in human B-ALL including Tbl1xr1. Together, these data indicate that a decrease in the normal transcriptional level of Pax5directly contributes to the development of B-progenitor ALL, consistent with Pax5 functioning as a haploinsufficient tumor suppressor. Next-generation sequencing to comprehensively identify all genes targeted by retroviral insertions, coupled with genome-wide copy number analysis, are in progress and should provide invaluable information on the range of mutations that cooperate with Pax5 haploinsufficincy to induce ALL.