Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion.

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.

Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells.

Canine bone marrow stromal cells were expanded to numbers in excess of 10(9) cells from the initial 10-20 ml of marrow aspirates and transfected to express high levels of human growth hormone (hGH) in vitro. Ex vivo-modified marrow stromal cells were used in a gene therapy model system for the systemic delivery of transgene products in dogs. Adherent bone marrow stromal cell cultures, established and expanded from iliac crest marrow aspirates from each of 8 dogs, were transfected with a hGH gene plasmid expression vector and shown to express from 0.54-3.84 micrograms/10(6) cells per 24 hr hGH in vitro. The transfected plasmid vector does not possess a eukaryotic origin of replication nor does it possess sequences required for efficient integration into the host cell genome. As such, expression was expected to be transient. Transfected cells were autologously reintroduced into each dog by either infusion into a foreleg vein or directly into iliac crest marrow. In two cases, the stromal cells were cryopreserved following transfection, and subsequently thawed and infused. In one case, the expanded stromal cells were first cryopreserved, and then thawed, recultured, transfected, and infused. Reintroduced cell numbers ranged from 2.2 x 10(7) to 2.6 x 10(9), with total hGH expression capacities ranging from 62 to 1,400 micrograms/24 hr. Plasma of each of the dogs contained detectable hGH for a mean of 3.1 days (SD +/- 0.8 day) ranging from 2 to 5 days following reinfusion of cells. Peak plasma levels ranged from 0.10 to 1.76 ng/ml. Similar hGH expression values, based upon total expression capacity of the cells infused and dog body weight, were obtained for all dogs. Vector-modified stromal cells were detectable, by polymerase chain reaction (PCR) analysis, in the peripheral circulation following reinfusion in all 4 dogs analyzed. In 3 of the dogs, modified stromal cells were detected for 8.5-15 weeks. In addition, modified stromal cells were detected in iliac crest marrow of 2 dogs for 9 and 13 weeks, respectively, following reinfusion. In another experiment, cultured bone marrow stromal cells were transfected with a human factor IX (hFIX) plasmid vector. Modified cells (5.57 x 10(8)), with a total hFIX expression capacity of 281 micrograms/24 hr, were reinfused, resulting in detectable hFIX in plasma continuously for 9 days with a peak level of 8 ng/ml on day 1. These results demonstrate that the ex vivo bone marrow stromal cell system is a potentially powerful method by which to deliver secreted transgene product to the systemic circulation of large animals.

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.