TREATMENT OF COMPLEMENT-MEDIATED DISORDERS

Abstract

Methods of treatment of complement-mediated disorders, in particular disorders associated with over-activity of the complement C3b feedback cycle (for example, age-related macular degeneration (AMD)), using gene therapy is described. According to the methods, levels of complement Factor I are elevated by administration of a recombinant viral vector encoding Factor I such that a therapeutically effective amount of the encoded Factor I is expressed from the vector in the subject. Recombinant viral vectors encoding Factor I, recombinant virus particles encapsidating the vectors, and their use in the methods of treatment, is also described.

Claims

1. A method for preventing, treating, or ameliorating a complement-mediated disorder in a subject in need thereof, which comprises administering to the subject a recombinant viral vector comprising nucleic acid encoding Factor I, or a fragment or derivative thereof that retains C3b-inactivating and iC3b-degradation activity, such that a therapeutically effective amount of the encoded Factor I, or the fragment or derivative thereof, is expressed from the nucleic acid in the subject, thereby increasing the level of C3b-inactivating and iC3b-degradation activity in the subject.

2. A method according to claim 1, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that exceeds a normal level.

3. A method according to claim 1 or claim 2, wherein the subject is administered with a recombinant virus particle that encapsidates the recombinant viral vector.

4. A method according to claim 3, wherein the recombinant virus particle infects the liver of the subject following administration, resulting in expression of the Factor I, or the fragment or derivative thereof, from the liver.

5. A method according to claim 3 or 4, wherein the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle encapsidating a rAAV vector.

6. A method according to claim 5, wherein the rAAV particle is pseudotyped to confer liver tropism.

7. A method according to claim 5 or 6, wherein the rAAV particle comprises one or two AAV2 ITRs, or derivatives thereof wherein each derivative AAV2 ITR comprises nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical over its entire length with the nucleotide sequence of a naturally occurring AAV2 ITR, and wherein the rAAV particle is pseudotyped with AAV8 capsid protein (rAAV2/8), or AAV2 pseudotyped with AAV9 capsid protein (rAAV2/9), or a derivative thereof comprising amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the amino acid sequence of a naturally occurring AAV8 or AAV9 capsid protein.

8. A method according to any preceding claim, wherein the recombinant virus particle is administered intravenously to the subject.

9. A method according to any preceding claim, wherein the recombinant viral vector is a non-integrating, episomal viral vector.

10. A method according to any preceding claim, wherein the encoded Factor I, or the fragment or derivative thereof, is expressed from a liver-specific promoter, such as a human alpha-1-anti-trypsin (hAAT) promoter.

11. A method according to any preceding claim, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is up to twice the normal level.

12. A method according to any preceding claim, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is up to 80%, or up to 60%, above the normal level.

13. A method according to any preceding claim, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is up to 40%, or up to 20% above the normal level.

14. A method according to any preceding claim, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is at least 5%, 10%, 15%, 20%, or 25% above the normal level.

15. A method according to any preceding claim, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is the level in serum of the subject.

16. A method according to any preceding claim, wherein the normal level of C3b-inactivating and iC3b-degradation activity in the subject is equivalent to that provided by 30-40 g/ml Factor I in serum of the subject.

17. A method according to any preceding claim, wherein the complement-mediated disorder is a disorder associated with over-activity of the complement C3b feedback cycle.

18. A method according to any preceding claim, wherein the complement-mediated disorder is age-related macular degeneration (AMD) (particularly early (dry) AMD, or geographic atrophy), dense deposit disease (DDD), atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathies, membranoproliferative glomerulonephritis Type 2 (MPGN2), atherosclerosis, chronic cardiovascular disease, Alzheimer's disease, systemic vasculitis, paroxysmal nocturnal haemoglobinuria (PNH), inflammatory or autoinflammatory diseases of old age, membranoproliferative glomerulonephritis type I (MPGN type I), membranoproliferative glomerulonephritis type III (MPGN type III), Guillain-Barre syndrome, Henoch-Schonlein purpura, IgA nephropathy, or membranous glomerulonephritis.

19. A method according to claim 18, wherein the subject is at risk of developing AMD.

20. A method according to claim 19, wherein the subject is homozygous or heterozygous susceptible for one or more SNPs associated with AMD.

21. A method according to claim 19 or 20, which further comprises determining whether the subject is at risk of developing AMD.

22. A method according to claim 21, wherein it is determined whether the subject is at risk of developing AMD by determining whether the subject is homozygous or heterozygous susceptible for one or more SNPs associated with AMD.

23. A method according to claim 20 or 22, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is at least 10% above the normal level if the subject is heterozygous susceptible for one or more SNPs associated with AMD.

24. A method according to claim 20 or 22, wherein the level of C3b-inactivating and iC3b-degradation activity in the subject is increased to a level that is at least 50% above the normal level if the subject is homozygous susceptible for one or more SNPs associated with AMD.

25. A method according to any preceding claim, which further comprises determining the level of C3b-inactivating and iC3b-degradation activity in the subject at least a week after the administration, and repeating the administration if the level of activity is found to be at, or below the normal level.

26. A method according to any preceding claim, wherein the Factor I is human Factor I with an amino acid sequence of SEQ ID NO: 2 or 4.

27. A method according to any preceding claim, wherein the fragment or derivative of Factor I is a polypeptide that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity across its entire length to human Factor I with an amino acid sequence of SEQ ID NO: 2 or 4.

28. A method according to any preceding claim, wherein the subject is a human subject.

29. A recombinant viral vector which comprises nucleic acid encoding Factor I, or a fragment or derivative thereof that retains C3b-inactivating and iC3b-degradation activity.

30. A recombinant viral vector according to claim 29, which is a non-integrating, episomal viral vector.

31. A recombinant viral vector according to claim 29 or 30, wherein the nucleic acid encoding Factor I, or the fragment or derivative thereof, is operably linked to a promoter.

32. A recombinant viral vector according to claim 31, wherein the promoter is a liver-specific promoter, such as a human alpha-1-anti-trypsin (hAAT) promoter.

33. A recombinant viral vector according to any of claims 28 to 31, which is a recombinant adeno-associated virus (rAAV) vector.

34. A recombinant viral vector according to claim 33, which comprises an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises the nucleic acid encoding Factor I, or the fragment or derivative thereof, operably linked to a promoter and a polyadenylation recognition site.

35. A recombinant viral vector according to claim 34, wherein the ITRs are AAV2 ITRs, or derivatives thereof, wherein each derivative AAV2 ITR comprises nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical over its entire length with the nucleotide sequence of a naturally occurring AAV2 ITR.

36. A recombinant virus particle, which comprises a viral capsid encapsidating a recombinant viral vector according to any of claims 29 to 35.

37. A recombinant virus particle according to claim 28 or 29 which is capable of transducing liver cells, particularly hepatocytes.

38. A recombinant virus particle according to claim 36 or 37, which is a rAAV particle.

39. A recombinant virus particle according to claim 38, wherein the rAAV particle is pseudotyped to confer liver tropism.

40. A recombinant virus particle according to claim 38 or 39, wherein the rAAV particle comprises AAV8 or AAV9 capsid protein, or a derivative thereof comprising amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the amino acid sequence of a naturally occurring AAV8 or AAV9 capsid protein.

41. A recombinant virus particle according to claim 40, wherein the rAAV particle comprises one or two AAV2 ITRs, or derivatives thereof wherein each derivative AAV2 ITR comprises nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical over its entire length with the nucleotide sequence of a naturally occurring AAV2 ITR.

42. A pharmaceutical composition, which comprises: a recombinant viral vector according to any of claims 29 to 35, or a recombinant virus particle according to any of claims 36 to 41; and a pharmaceutically acceptable carrier, excipient, or diluent.

43. A pharmaceutical composition according to claim 42, which is suitable for intravenous administration.

44. A kit, which comprises: a recombinant viral vector according to any of claims 29 to 35, or a recombinant virus particle according to any of claims 36 to 41; and a pharmaceutically acceptable carrier, excipient, or diluent.

45. A kit for production of rAAV particles, which comprises: a rAAV vector according to any of claims 33 to 35; and one or more helper plasmids comprising nucleic acid encoding AAV replication and capsid proteins, and genes required for a productive AAV life cycle.

46. A kit according to claim 45, which comprises a first helper plasmid comprising the nucleic acid encoding AAV replication and capsid proteins, and a second helper plasmid comprising the nucleic acid encoding genes required for a productive AAV life cycle.

47. A recombinant viral vector according to any of claims 29 to 35, a recombinant virus particle according to any of claims 36 to 41, or a pharmaceutical composition according to claim 42 or 43, for use as a medicament.

48. A recombinant viral vector according to any of claims 29 to 35, a recombinant virus particle according to any of claims 36 to 41, or a pharmaceutical composition according to claim 41 or 42, for use in the treatment of a complement-mediated disorder.

49. Use of a recombinant viral vector according to any of claims 29 to 35, a recombinant virus particle according to any of claims 36 to 41, or a pharmaceutical composition according to claim 42 or 43, in the manufacture of a medicament for the treatment of a complement-mediated disorder.

50. A vector, particle, or composition according to claim 48, or use according to claim 48, wherein the complement-mediated disorder is a disorder associated with over-activity of the complement C3b feedback cycle.

51. A vector, particle, or composition according to claim 50, wherein the complement-mediated disorder is age-related macular degeneration (AMD) (particularly early (dry) AMD, or geographic atrophy), dense deposit disease (DDD), atypical haemolytic uraemic syndrome (aHUS), C3 glomerulopathies, membranoproliferative glomerulonephritis Type 2 (MPGN2), atherosclerosis, chronic cardiovascular disease, Alzheimer's disease, systemic vasculitis, paroxysmal nocturnal haemogtobinuria (PNH), inflammatory or autoinflammatorys disease of old age, membranoproliferative glomerulonephritis type I (MPGN type I), membranoproliferative glomerulonephritis type III (MPGN type III), Guillain-Barre syndrome, Henoch-Schonlein purpura, IgA nephropathy, or membranous glomerulonephritis.

52. A vector, particle, or composition according to claim 50, wherein the complement-mediated disorder is age-related macular degeneration (AMD).

Description

[0211] Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

[0212] FIG. 1 shows an outline of the steps of complement activation;

[0213] FIG. 2 shows the feedback loop of the alternative pathway of vertebrate complement;

[0214] FIG. 3 shows the proteolysis of C3 during complement activation. C3 consists of the a and chain. The -chain is not modified while the a-chain is cleaved several times: i) C3a is cleaved off by a C3 convertase and the remainder protein is now called C3b; ii) Fl cleavage at two sites, releasing the C3f peptide (FH as co-factor), it is now called iC3b or C3bi; iii) with CR1 as co-factor, Fl cleaves again in the 68 kDa fragment of iC3b. While C3c diffuses off. C3dg stays attached to the cell surface and can be cleaved further by trypsin-like proteases;

[0215] FIG. 4 shows a schematic representation of AAV gene therapy;

[0216] FIG. 5 shows an AAV2/8 construct used for over expression of serum levels of Factor I. The transgene, here Factor I, is inserted between a promoter and a polyadenylation recognition site. Flanking on both sides are two inverted terminal repeats (ITR). The capsid sequence and further adenoviral genes are packed into other plasmids;

[0217] FIG. 6 shows elevated Factor I levels measured by western blot. Mouse serum was diluted to 5% and 10 I were separated by SDS PAGE. Fl was detected with an a-FI antibody (1:500) that reacts with the heavy chain of mFI. The experiment was performed twice;

[0218] FIG. 7 shows representative results from an inhibition ELISA. (a) The assay is calibrated with known concentrations of purified recombinant mFI. Concentrations lower than 0.25 g/ml give a positive signal, (b) Normal mouse serum (NMS) is positive at concentrations lower than 0.625%. (c-f) Inhibition ELISA using sera from mice injected with AAV-constructs. Only one serum sample per group is shown here. The experiment was performed twice in triplicate;

[0219] FIG. 8 shows the results of a C3b and iC3b in vitro cleavage assay to measure functional activity of over-expressed Factor I. Substrate, i.e. C3al chain, is generated quickly and the rate of its breakdown is compared, i.e. generation of C3dg. C3 cleavage was detected with a-hC3dg (1 g/ml). The experiment was performed twice; and

[0220] FIG. 9 shows the results of an iC3b deposition assay to test functional activity of over-expressed Factor I. Serum of injected mice was diluted to 25% in alternative pathway complement fixation buffer and loaded onto an LPS-coated plate. After incubation for 1 hour at 37 C., bound iC3b was detected with an a-human C3c antibody. Absorption was measured at 415 nm. The experiment was performed twice in duplicate.

EXAMPLE 1

AAV-Expression System

[0221] This example describes preparation of a recombinant viral vector encoding murine Factor I that enables over-expression of Factor I in murine hepatocytes, and preparation of viral particles (virions) encapsidating the vector.

[0222] An adeno-associated virus (AAV) construct was used. It consists of an AAV2 viral backbone that was pseudotyped with the AAV8 capsid protein in order to confer liver tropism. The virus, therefore, mainly infects the liver, and the Fl transgene is over-expressed at its natural site. To further suppress extra-hepatic expression of Factor I transgene, an -1-anti-trypsin (AAT) promoter with two additional ApoE hepatic control regions was used (see FIG. 5). All required genes for virus production are split up between three plasmids that are co-transfected into a cell line that provides the remaining missing genes for AAV packaging, as further described below.

[0223] AAV (wild type): The 4.7 kb genome of wildtype AAV is characterized by two inverted terminal repeats (ITRs) and two sets of open reading frames (ORFs), which encode the replication (Rep) and capsid (Cap) proteins. The Rep ORFs encode four proteins (78, 68, 52, and 40 kDa), which function mainly in regulating AAV replication and integration. The Cap ORFs encode three structural proteins (85 kDa (VP1), 72 kDa (VP2) and 61 kDa (VP3)). VP1:VP2:VP3 ratios are approximately 1:1:8 or 1:1:10 in the capsid.

[0224] Recombinant AAV (rAAV): rAAV used in this example is AAV2 pseudotyped with AAV8 capsid (rAAV2/8) to confer liver tropism. The rAAV2/8 virions are packaged in HEK293 cells by triple transfection using the three plasmids described below: [0225] 1. pAM2AA (ITRs and transgene expression cassette): In this plasmid the 145 bp inverted terminal repeats (ITRs) from AAV2 flank the transgene cassette. The two ITRs are the only cis elements essential for all steps in the AAV life cycle. They function as the origin of DNA replication, provide packaging and integration signals, and serve as regulatory elements for WT AAV gene expression. The pAM2AA cassette includes the human a-1-antitrypsin (hAAT) promoter with two ApoE enhancers followed by the cDNA encoding the transgene. The 3-untranslated region (UTR) contains the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGH polyA). The packaging capacity of WT AAV is approximately 4.7 kb. In pAM2AA, the regulatory regions take up * 2390 bp, leaving * 231 Obp for insertion transgenes. [0226] 2. p5E18VD/8 (AAV helper sequences): The deleted viral coding sequences from AAV2, including Rep and Cap genes are present in this plasmid driven by the p5 AAV promoter. Here, the sequences encoding AAV2 capsid are replaced by sequences encoding AAV8 capsid. There is no homology between vector and helper sequences, reducing the possibility of generating wild type AAV recombinants. [0227] 3. pXX6 (adenoviral helper functions): The adenoviral genes essential for a productive AAV life cycle are the E1a, E1b, E2a, E4 and VA RNA genes. E1a serves as a transactivator which upregulates transcription of adenoviral genes as well as the AAV Rep and Cap genes. E1b interacts with E4 to facilitate transportation of viral mRNAs. E4 is also involved in facilitating AAV DNA replication. E2a and VA RNA act to enhance the viral mRNA stability and efficiency of translation especially for the Cap transcripts. pXX6 contains the essential adenoviral helper genes but lacks adenovirus structural and replication genes. Essential helper genes included in pXX6 are E2a, E4 and VA genes. Both E1a and E1b have been deleted, and the missing E1a and E1b genes are complemented by HEK293 cells.

Preparation of Recombinant AAV Vector Encoding Murine Factor I (AAV mFH

[0228] To enable cloning of Factor I into the pAM2AA expression vector, the restriction sites had to be changed into compatible enzyme recognition sites (using primer sequences: FI-AAV-F: 5-TCT AGA GGA TCC GCC ACC ATG AAG CTC-3 (SEQ ID NO: 5); and FI-AAV-R: 5-AAG CTT GGC GGC CGC TCA GAC ATT GTG TTG AGA AAC AAG AGA CCT TC-3 (SEQ ID NO:6). The new sequences were attached to the mouse Factor I cDNA via PCR amplification. After purification of the amplified construct, it was Ikjated into pAM2AA. The cDNA of murine complement Factor I was cloned into pAM2AA using Xbal and Hindlll restriction sites. Once the vector had been prepared, an AAV_GFP vector was treated the same way alongside all subsequent virus packaging and purification steps. This ensures that no errors occurred since expression of hepatic GFP can easily be tracked visually under a fluorescence microscope.

Preparation of rAAV2/8 Virions

[0229] The pAM2AA vector with cDNA of murine complement Factor I was transformed into SURE2 cells to prevent unwanted recombination events. HEK293 cells were transfected with all three plasmids using CaPO*. Two days after transfection, cells were harvested and cells were lysed by repeated freeze-thawing in a dry ice/100% ethanol bath and then stored at 80 C. AAV virions were purified on a cesium chloride gradient and virus particles were quantified by real-time PCR.

EXAMPLE 2

In Vivo Overexpression of Complement Factor I Fay an Adeno-Associated Virus Delivery System

[0230] This example describes injection of mice with virions encapsidating the AAVjmFI vector produced as described in Example 1, determination of plasma levels of Factor I following injection of the virions, and an assessment of the down-regulation of the alternative complement pathway in the mice following injection.

Injection of Mice

[0231] 12 mice were divided into 4 groups, which received different concentrations of AAVjnFI or the control virus preparation: [0232] tow mFI=510.sup.9 virions [0233] med mFI=510.sup.10 virions (=100% liver transduction in previous experiments) [0234] high mFI=510 virions [0235] GFP=510 virions

[0236] The mice were injected intravenously into the tail vein. Blood was taken after 4 weeks, and after 8 weeks the mice were culled and their serum was analysed.

Results

Quantification of Elevated Factor I Levels

[0237] To quantify Fl levels, a polyclonal custom-made a-mFI antiserum was ordered. In the meantime, a western blot was performed. Here, a-FI (Santa Cruz, sc-69465) was used as detecting antibody to roughly quantify Fl levels. Serum was diluted in sequential steps to reduce pipetting errors. The relative concentration of Fl was determined as follows: AAV_GFP<AAV.FI low<AAV_FI medium<AAV_FI high (see FIG. 6).

[0238] Once the antiserum arrived, a much more sensitive assay, an inhibition ELISA, was performed. Serum was pre-incubated with a before-determined concentration of a-mFI-antiserum and then incubated on a microtiter plate coated with recombinant mFI. Preparation and binding specificity of the a-mFI-antiserum are described in the Materials and Methods section below. Only unbound antibodies are able to react with the immobilised antigen on the plate. Therefore, the assay only gives a positive result if the amount of a-mFI-antibodies in the antiserum exceeds the mFI present in serum. The assay is calibrated with known Factor I concentrations.

[0239] It was found that antiserum concentrations above 75 g/ml give a strong positive signal when the microtiter plate is coated with 1 g mFI per well (not shown). FIG. 7 shows representative results from an inhibition ELISA. The assay gives a positive signal at concentrations lower than 0.25 g/ml (FIG. 7a). Using this calibration, the concentration of Fl in a serum sample can be determined. First, a normal sample of wild-type mouse was tested and concentrations lower than 0.625% gave a positive signal (FIG. 7b). Knowing that the threshold of a positive signal in this assay is 0.25 g/ml mFI, the concentration of mFI in 100% serum can be calculated, i.e. 40 ug/ml. The same calculation was done for each of the mouse sera injected with the AAV-constructs (FIG. 7c-f), and the concentration range per group was determined (see Table 2).

TABLE-US-00003 TABLE 2 Quantification of Factor I after over-expression by an adeno-associated virus expression system Group FI g/mL FI increase AAV_GFP 20-40 1x AAV_FI low 40-80 1-2x AAV_FI medium 80 2x AAV_FI high 80-160 2-4x

[0240] Starting from 20-40 Mg/ml Fl in the GFP control group, the concentration is 40-80 Mg/ml in the low-, 80 g/ml in the medium- and 80-160 Mg/ml Fl in the high Fl dose. It was concluded that the serum concentration can be raised up to four times normal levels by AAV gene therapy.

Functional Analysis of Serum with Elevated Factor I Levels

[0241] The serum of the transgenic mice was analysed in a similar manner as the recombinant Factor I. Since the over-expressed protein will, theoretically, be processed and secreted as endogenous Fl, the sera of the mice could be directly compared with each other. After Fl increase has been confirmed by immunoblotting and by an inhibition ELISA, the functional activity of the over-expressed enzyme had to be confirmed. Therefore, the sera were analysed for Fl functional activity by an in vitro C3b cleavage assay, a hemolysis assay and an iC3b deposition assay.

[0242] C3b and iC3b cleavage was measured in an in vitro assay. Human C3 was digested to C3b with trypsin and incubated with hFH and serum from transgenic mice. After 30 minutes, 1 \ig human C3 was loaded onto a SDS gel and blotted with clone 9. C3b is first cleaved into iC3b in a quick reaction (the antibody reacts with the C3ctl chain of iC3b) and then further into C3dg in a much slower reaction. This second reaction can be speeded up by increased Fl concentrations. It is shown that human C3b incubated with human Factor H and serum from the different mouse groups was more efficiently cleaved to C3dg within the incubation period in the AAV_FI medium and AAV_FI high group (see FIG. 8, lanes 10-14). Mice that have been injected only with a control plasmid show almost no C3dg band within the incubation period.

[0243] The next step was to show that over-expressed Factor I was also functionally active in whole serum. For this, a microtiter plate was coated with LPS and C3b/iC3b deposition was measured after incubation with transgenic mouse serum at 37 C. The results are shown in FIG. 9. As with the recombinant protein, mice injected with AAV_mFI showed less C3b/iC3b deposition on LPS since their positive feedback loop of the alternative pathway was interrupted by Factor I. C3b/iC3b deposition was reduced by 11% in the AAVJow Fl, by 38% in the AAV_medium Fl and by 50% in the AAV % high Fl group.

[0244] It was concluded that complement Factor I can be over-expressed by gene therapy, using a viral vector, to levels which cause significant down-regulation of the alternative complement pathway.

Discussion

[0245] Gene therapy is a new and exciting therapeutic option for the treatment of various diseases. It enables in vivo expression of proteins after transfection of the respective cell with a construct harbouring the DNA sequence of the protein of interest. Theoretically, gene therapy can be utilised for expression of every protein, although practically, there are usually limitations, such as for example the length of the sequence. Using gene therapy, missing proteins can be replaced. However, this always carries a risk of immunogenicity. We have appreciated that this risk does not occur if concentrations of existing (endogenous) proteins are raised. In this study we wanted to elucidate whether complement Factor I can be over-expressed by gene therapy to levels which cause significant down-regulation of the alternative complement pathway.

[0246] We have succeeded in generating a vector for transgenic expression of mouse complement Factor I. The viral expression system used is based on AAV2 and its capsid is derived from AAV8. Together this AAV2/8 construct mainly results in liver transduction in mice, although virus particles can be also found in extra-hepatic tissue. To increase liver-specific expression, the transgene is under the control of the -1-anti-trypsin promoter with two ApoE hepatic control regions for high-level and specific expression in hepatocytes. Therefore, transgene expression should only occur in hepatocytes (although, sometimes there can be very little transgene expression also in pancreatic tissue, which is a very closely related tissue).

[0247] We have demonstrated that over-expression of Fl by the AAV expression system used clearly has an effect on complement down-regulation and secondly that this effect is titrate-able and becomes more profound as the vector dose is increased. In order to measure the total Fl increase, a polyclonal antibody against mouse Factor I was raised in a rabbit, since commercially available a-FI antibodies were found unsuitable for measuring mouse Fl concentrations in an ELISA (they do not react with native Fl but only with the denatured protein in a western blot). Using this polyclonal custom-made antibody it was possible to show that serum concentrations were increased up to four times normal levels, i.e. from 20-40 g/ml in control mice to 80-160/I Fl in transgenic mice.

[0248] Another interesting question is whether transgene expression is further increased in an acute phase reaction. Since in the construct used, Fl is under the control of the -1-anti-trypsin promoter (a-1-anti-trypsin and Fl are both positive acute phase reactants), it can be expected that transgene expression will also be increased in an acute phase reaction. This could be tested in a straightforward animal experiment that could be easily performed by injection of LPS or IL6 into wildtype and transgenic mice and then compare the rate of Fl increase. Assuming both endogenous and transgenic Fl expression would increase, then this could be a solution to reduce the initial virus load during AAV administration but with the option to increase expression if required.

[0249] Factors such as host immunity have prevented the widespread use of AAV in man. Once exposed to AAV, people develop neutralizing antibodies that block gene delivery. However, by engineering of the capsid proteins, new variations can be generated that have higher transduction efficiencies and are not recognised by neutralising antibodies. New approaches to limit these immune responses are therefore undertaken (Mingozzi, et al., Science Translational Medicine, 5(194):194ra92-194ra92, July 2013). Recent clinical studies have shown success of a single infusion of an AAV vector leading to two years of therapeutic levels of Factor IX in men with severe hemophilia B (Mingozzi and High, Blood, 122(1):23-36, July 2013), although it should be pointed out that an increase of only 1% of normal FIX levels, substantially ameliorates the severe bleeding phenotype in hemophilia B patients (Nathwani, et al., N. Engl. J. Med., 371 (21): 1994-2004, November 2014). Before, AAV transgene expression of FIX in skeletal muscle was shown to persist for 10 years, although in this case, circulating FIX levels remained subtherapeutic (<1%) (Buchlis, et a/., Blood, 119(13):3038-3041, March 2012).

[0250] An increase of Fl by 50% maximal is what is aimed for in human therapy. This is by far exceeded in mice using the AAV_mFI-construct, and the iC3b deposition assay shows a reduction of up to 50% less C3b deposition at the high AAV_mFI dose. Separately, we have shown that 50% more Fl converts the activity of an high-risk complotype to the activity of a low-risk complotype (Lay er al., 2015 (supra)). Since both of these complotypes are extremely rare and the majority of people will be within both extremes, an effect for risk reduction for the majority of people is only required within the range of 50% Fl increase.

Materials and Methods

[0251] q-Mouse Factor I

[0252] This polyclonal antibody is commercially available from Santa Cruz Biotechnology, Inc (# sc-69465). The antibody was raised in goats against a peptide in the heavy chain of mouse Factor I and recognizes the heavy chain in reducing and non-reducing western blots or ELISAs. If still intact, the antibody also recognises the pro-enzyme. Nevertheless, the antibody is not precipitating because the peptide used for immunisation of is too short.

[0253] Normal working concentration 1.500.

g-Mouse Factor I Antiserum

[0254] This antibody is not commercially available and was ordered from Absea Biotechnology Ltd. in order to get a precipitating antibody to mFI which would allow easy determination of Factor I levels in a Ouchteriony double immunodiffusion assay. A rabbit was immunised with a peptide fragment (203aa-510aa) of recombinant mouse Factor I that was produced in bacteria. Once tested, the antibody turned out to be non-precipitating and therefore, an inhibition ELISA was developed to measure the levels of Factor I in serum. To get a multivalent antibody to mouse Factor I that precipitates Fl, Factor I purified from mammalian cell culture was sent to Absea Biotechnology Ltd. but the immunisation period (8 weeks) for this second a-mouse Factor I antiserum was not completed in time but can be used for future experiments.

Clone 9 Antibody

[0255] Clone 9 is a rat a-human C3g antibody that recognises a neo-epitope in C3g that only becomes accessible if C3 is cleaved to iC3b or C3dg by Factor I (Lachmann, et al., Immunology, 41(3):503-515, November 1980). Under native conditions, clone 9 only reacts with iC3b or C3dg of human origin, whereas under denaturing conditions it detects the a, a chain and the 68 kDa fragment of human C3 because all these three fragments include its epitope in C3g. Normal working concentration for detection in a western blot is 0.5 g/ml and as capture antibody for coating is 1.35 Mg/ml. The names clone 9 and alpha-hCZq antibody will be used interchangeably.

Clone 4 Antibody

[0256] Clone 4 is a rat monoclonal anti-C3c antibody that recognizes a conformational epitope in C3c and reacts with C3, C3b, iC3b and C3c (Lachmann, et al., Immunology, 41(3):503-515, November 1980). It therefore does not bind to Cdg or C3g because of the absence of the epitope in C3c. Normal working concentration is 5 g/ml.

g-Human C3c

[0257] This antibody is commercially available from Dako and is used to detect C3b and iC3b deposition. It is used at a concentration of 1:5000 and detected with an a-rabbit secondary antibody.

Enzyme-Linked Immunosorbent Assay (ELISA)

[0258] iC3b deposition assaydone 9 assay* Maxisorb Nunc Plates were coated overnight at 4 C. with clone 9 at a concentration of 1.25 g/well in coating buffer. Next day, the plates were washed 5 with washing buffer (=PBS-0.05% Tween 20) and blocked with 4% Marvel milk powder in PBS-T for 2 hours at room temperature. The plates were washed 5 and diluted serum samples (1:2000 in PBS/gelatin) were added to each well (preparation of serum samples is described below). After a 1 hour incubation, the plate was washed and incubated for 1 hour with biotinylated clone 4 at a concentration of 5 g/ml in PBS/gelatin. The plates were washed 5 and incubated with 1:1000 Extravidin-HRP in PBS/gelatin. Finally, after extensive washing, 100 I TMB substrate (Invitrogen) were added and plates were incubated on a plate shaker for 30 minutes. The reaction was stopped with 50 I H2SO4 and the plates were read on a Microplate reader (450 nm).

[0259] C3b deposition assay Maxisorb Nunc Plates were coated overnight with mannan (Sigma, mannan from Saccharomyces cerevisiae) or lipopolysaccharide (Sigma) Ig/well at 4 C. in coating buffer. On the next day, the plates were washed in TBS-0.05% Tween-20 and blocked with 1% BSA in TBS-T at room temperature. After 2 hours, the plates were washed and incubated with serial dilutions of recombinant proteins and/or sera. After a 1 hour incubation at 37 C., plates were washed with TBS-T and incubated for 1 hour with polyclonal rabbit a-human C3c complement (Dako), used 1:5000 in TBS-T. After extensive washing, a-rabbit IgG (whole molecule)-alkaline phosphatase (produced in goat, Sigma) 1:5000 in TBS-T was added for 1 hour. The plates were washed 4 with TBS-T and the assay was developed using Fast p-Nitrophenyl Phosphate Tablets (Sigma) and measured (405 nm). Alternatively, the assay development was stopped by addition of 3 M NaOH and read afterwards.

[0260] Inhibition ELISA In an inhibition ELISA, microtiter plates are coated with antigen and, after blocking, the concentration of antiserum is determined that gives a strong signal when detected with the secondary antibody. This concentration is then used in the inhibition assay and mixed with serial dilutions of a sample with an unknown antigen concentration. At the same time, a dilution series of a known antigen concentration is prepared. During this pre-incubation step, immune complexes form and when the mix is loaded on the coated microtiter plate, only samples in which the concentration of antigen does not exceed the amount of specific antibody give a positive signal. If there is excess antigen, no free antibodies are available that can bind to the immobilised antigen on the microtiter plate. Therefore, a positive result in an inhibition assay shows the dilution of sample in which the amount of free antigen is limiting and by comparison with the standard dilutions of known antigen concentrations, the unknown concentration can be determined.

[0261] For determination of the concentration of mouse Factor I in a serum sample, Maxisorb Nunc Plates were coated overnight at 4 C. with purified recombinant mouse Factor I (1 g/well) in coating buffer. Next day, plates were blocked with 1% BSA in TBS-T for 2 hours at room temperature. During this incubation, dilutions of mouse serum (5% down-wards) or purified mouse Factor I (2 Mg/ml downwards) were prepared in 1% BSA/150 mM NaCl. Next the dilutions were mixed 1:1 with the determined concentrations of purified and biotinylated a-mFI IgGs, i.e. 150 Mg/ml. The samples were incubated at room temperature for 1 hour on a plate shaker and then loaded onto the coated plate. After one hour incubation, the plate was extensively washed and bound biotinylated a-Factor I antibody was detected with Extravidin-alkaline phosphatase at a concentration of 1:5000. The plates were washed 4 with TBS-T and the assay was developed using Fast p-Nitrophenyl Phosphate Tablets (Sigma) and measured (405 nm).

Western Blot

[0262] Western blot analysis was performed to detect specific proteins or to confirm their presence in a sample. First, gel electrophoresis was performed to separate the proteins according on their size. The protein samples were boiled up in 4 loading buffer to denature them; -Mercaptoethanol was added if reduced protein was required for analysis. SDS gel electrophoresis was performed on a 4-12% bis-tris protein gel (Invitrogen) for 50 minutes at 200 V. After separating the proteins, a wet transfer was performed to transfer the separated proteins onto a PVDF membrane where target proteins can detected with specific antibodies.

[0263] The blot was assembled by stacking in transfer buffer-equilibrated blotting paper, gel and membrane (first activated with MeOH) into a transfer cassette. The transfer was performed for 1 hour at 350 mA, 60 W.

[0264] Next, the membrane was blocked for 30 minutes on a rotator in blocking buffer. Primary and secondary antibody were both incubated for 1 hour rolling, in between which the membrane was washed 35 minutes in wash buffer. The last washes were done in TBS-T, pH 8, because both used substrates gave stronger signals when the last washes have been in slightly alkaline pH. Depending on the conjugate of the secondary antibody, different substrates were used: proteins were detected chromogenically by addition of 3 ml of TMB or AP substrate (Life Technologies) or, in case of peroxidase conjugated antibodies were also detected chemiluminescently after addition of ECL reagent.

Preparation of Serum

[0265] Serum preparation was performed as described in (Lachmann, J. Immunol. Methods, 352(1-2): 195-197, January 2010). Blood was taken under sterile conditions and left to clot at room temperature. The initial centrifugation is usually carried out in a bench centrifuge at about 3.000g for about 5-10 minutes at room temperature. The serum is taken and a second, high speed, centrifugation at; 20.000g for 2-5 minutes is carried out. This step is essential to remove all fragments of RBCs that can later distort the results. The serum removed from this second centrifugation can then be chilled, aliquoted and frozen for future experiment. It should be noted that serum used for functional assays should never be stored at temperatures above 80 C. Repeated freeze/thawing should be avoided by preparation of aliquots.

In Vitro Assays

[0266] Furin digest of pro-Factor I Both, Hek and CHO cells, were secreting only partially processed mFI and the majority was the inactive pro-enzyme of Factor I. In order to get active Factor I, the purified pro-enzyme was digested with the protease furin. In brief, purified mFI was dialysed against TBS and then adjusted to 10 mM CaCl.sub.2). 1 unit of furin was added per 25 g mFI. The reaction was carried out overnight at 30 C. On the next day, Factor I was used immediately or aliquoted and stored at 80 C.

[0267] C3b cleavage assay In a C3b cleavage assay, C3b was first prepared by limited tryptic digest as described in (Bokisch, et al., J. Exp. Med., 129(5):1109-1130, May 1969). In brief, 20 human C3 were incubated with 0.60 trypsin (10 g/ml) for exactly 60 seconds at 20 C. The reaction was stopped by addition of 2.4 I soy-bean trypsin inhibitor (10 g/ml). This limited digest results in generation of C3b of C3 by cleaving off C3a. Next, C3b was mixed with various amounts of murine Factor I or human Factor I and 2 I human Factor H and the reaction volume was completed to 100 with TBS. Alternatively, murine serum was added as a source of Factor I. The reaction was carried out for 30-60 minutes at 37 C. and then stopped by addition of reducing 4 loading buffer and boiling. The samples were either analysed by western blot or stored at 4 C.

[0268] Time course In order to test the ability of recombinant mouse Factor I to cleave C3b and iC3b, a time course assay was developed. For this, serum was thawed rapidly at 37 C., vortexed briefly, and then placed on ice. The serum was then mixed with recombinant human or mouse Factor I, and then zymosan was added to a final concentration of 5%. Alternative pathway buffer was used as buffer in this assay. Once prepared, the mixture was rotated in a 37 C. incubator and at selected time points 40 I of each sample were removed into 100 of 50 mM EDTA. The sampling times were: 0, 30, 60, 120, 180, 240, 480, 600 and 1440 minutes. At each time point the samples were microfuged to remove the zymosan and frozen at 80 C. until tested by ELISA. Alternatively, the assay was also done with LPS as complement activating reagent which was the advantage that it is soluble.

[0269] All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that any reference affects the novelty or non-obviousness of the embodiments described herein.