Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B

Abstract

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

Figure 1

Lines of hemophilia B mice. (a) Primary amino acid sequence of hF.IX and locations of F9 mutations expressed in transgenic lines of hemophilia B mice: late stop codon at amino acid residue 338 (“LS”, crm⁻, i.e., no circulating antigen); crm⁻ G381E missense mutation (“CH”, identical mutation as in hemophilia B dogs of University of North Carolina–Chapel Hill strain); crm⁺ R180W missense mutation. Boxed sequences contain CD4⁺ and CD8⁺ T-cell epitopes in C3H/HeJ mice. (b) Immunostain of hF.IX in livers of the four lines. Original magnification ×100.

Figure 2

Systemic expression of and humoral immune responses to hF.IX as a function of time after muscle-directed gene transfer in hemophilia B mice. (a–d) Coagulation times (aPTT in seconds). (e–h) Inhibitory antibody titers against hF.IX in BU. (i–l) IgG1 titers against hF.IX in ng/ml. (a,e,i) Mice with F9 gene deletion (“Null” mutation, n = 6). (b,f,j) Mice expressing F9 with late stop codon at amino acid residue 338 (“LS” mice, n = 5). (c,g,k) Mice expressing F9 with crm⁻ missense mutation G381E as found in the hemophilia B dogs of the Chapel Hill strain (“CH” mutation, n = 5). (d,h,l) Mice expressing F9 with crm⁺ missense mutation R180W (“MS” mutation, n = 6). Each line represents an individual animal. Horizontal lines for aPTT indicate range of normal mouse plasma (25–35 seconds) and of untreated hemophilia B plasma (>60 seconds). aPTT, activated partial thromboplastin times; BU, Bethesda unit; hF.IX, human factor IX, IgG1, immunoglobin G-1.

Figure 3

Systemic expression of and humoral immune responses to hF.IX as a function of time after liver-directed gene transfer in hemophilia B mice. (a–d) Coagulation times (aPTT in seconds). (e–h) Plasma levels of hF.IX (in ng/ml). (i–l) Inhibitory antibody titers against hF.IX in BU. (m–p) IgG1 titers against hF.IX in ng/ml. (a,e,i,m) Null mice (with F9 gene deletion, n = 4). (b,f,j,n) Mice expressing F9 with late stop codon at amino acid residue 338 (“LS” mice, n = 4). (c,g,k,o) Mice expressing F9 with crm⁻ missense mutation G381E as found in the hemophilia B dogs of the Chapel Hill strain (“CH” mutation, n = 4). (d,h,l,p) Mice expressing F9 with crm⁺ missense mutation R180W (“MS” mutation, n = 5). Each line represents an individual animal. Horizontal lines for aPTT indicate range of normal mouse plasma (25–35 seconds) and of untreated hemophilia B plasma (>60 seconds). aPTT, activated partial thromboplastin times; BU, Bethesda unit; hF.IX, human factor IX.

Figure 4

Summary of humoral immune responses to hF.IX after liver- or muscle-directed gene transfer or protein therapy in Null mice. (a) IgG anti-hF.IX and (b) inhibitor titers upon protein therapy in Null mice (intravenous injection of 1 IU recombinant hF.IX protein once per week starting at week 0, n = 4, dashed line) in comparison to muscle (dotted line) and liver (solid line) gene transfer to Null mice. hF.IX, human factor IX; IgG, immunoglobin G.

Figure 5

Immunofluorescent staining of hF.IX (red) and CD8 (green) in muscle and liver cross-sections after gene transfer to hemophilia B mice. (a–d) Muscle gene transfer to Null mice, (e) LS mice, (f) CH mice, and (g) MS mice. (c) Hematoxylin and eosin stain. Muscles were collected 30 days after gene transfer except for d (90 days). Two examples of the large number of CD8⁺ cells visible in a are indicated by arrows. (h–k) Hepatic gene transfer to Null mice (h, 1 month after gene transfer; i, 3 months, j–k, 2 months). DAPI stain of nuclei is shown in blue. Inserts: enlargement of CD8⁺ cell. Original magnification: a, ×40; b–g, ×200; h–k, ×100. DAPI, 4′-6-diamidino-2-phenylindole; hF.IX, human factor IX.

Figure 6

Identification of CD8⁺ T-cell epitope in C3H/HeJ mice by immunization using IM injection with Ad-hF.IX vector. (a) Matrix of pools of eight 15-mer peptides spanning the entire mature hF.IX amino acid sequence. Highlighted are pools 2 and 18, which yielded an IFN-γ⁺ CD8⁺ response, pointing toward peptide p74 to contain a CD8⁺ T-cell epitope. (b) Splenocytes showed an increased frequency of IFN-γ⁺ CD8⁺ when stimulated in vitro with peptide pools 2 and 18 and analyzed by flow cytometry. (c) Summary of IFN-γ⁺ CD8⁺ frequencies after stimulation with different peptide pools. (d–e) IFN-γ⁺ CD8⁺ T cell frequencies after stimulation with peptide p74, flanking peptides p73 or p75, mock media, an irrelevant peptide (ova), or nonspecifically stimulated with PMA or SEB. Experiments were performed in (b–d) wild-type or (e) F9^−/− (F9 gene deletion) C3H/HeJ mice. (f) IFN-γ⁺ CD8⁺ T cell frequencies after IM administration of AAV-2-CMV-hF.IX vector in F9^−/− C3H/HeJ mice and in vitro stimulation with irrelevant peptide (Ova), peptide p74, or F.IX protein. Shown are average frequencies ± SEM for three animals (experiments in b–d were performed using splenocytes pooled from three to four mice, while splenocytes from individual animals were assayed separately in the experiment in f; *P < 0.05, **P < 0.01 by unpaired Student's t-test). AAV-2, adeno-associated viral; CMV, cytomegalovirus; hF.IX, human factor IX; IFN-γ, interferon-γ.

Figure 7

ELISpot assays to determine frequencies of hF.IX-specific cytokine secreting cells in hemophilia B C3H/HeJ containing different F9 mutations. (a) IFN-γ producing splenocytes following muscle-directed gene transfer and in vitro stimulation with peptide p74 (CD8⁺ T-cell epitope, black bars), peptide 2A-54 (CD4⁺ T-cell epitope, gray bars), or hF.IX protein (hatched bars), or mock (white bars). Mice were assayed individually; stimulations were performed in triplicate for each antigen and mouse. Results are average ± SEM as SFU per million splenocytes (n = 3–4 per line; Null: F9 gene deletion, LS: R338stop, CH: G381E, MS: R180W). (b) IL-5 producing splenocytes following muscle-directed gene transfer and in vitro stimulation with peptide 2A-54 (CD4⁺ T-cell epitope), or hF.IX protein, or mock media (n = 3–4 mice per line). (c) IFN-γ producing IHL or splenocytes following liver-directed gene transfer in Null mice and in vitro stimulation with peptide p74 (CD8⁺ T-cell epitope), peptide 2A-54 (CD4⁺ T-cell epitope), or hF.IX protein, or mock media (n = 3). In contrast to splenocytes, IHL were pooled prior to culture, and data are reported for individual wells. (d) IL-5 producing IHL or splenocytes following liver-directed gene transfer in Null mice and in vitro stimulation with peptide peptide 2A-54 (CD4⁺ T-cell epitope), or hF.IX protein, or mock media. Examples of wells are shown below each graph. hF.IX, human factor IX; IFN-γ, interferon-γ; IHL, intrahepatic lymphocytes; SFU, spot-forming units.