WO2023079297A1

WO2023079297A1 - Mutant complement factor i variants with increased activity

Info

Publication number: WO2023079297A1
Application number: PCT/GB2022/052787
Authority: WO
Inventors: Fabrizio Comper; Samantha CORREIA; Jonathan Foley; Tudor ILCA; Azadeh Kia; Erald SHEHU; Sid SRIDHARAN; Daniel Verhoef
Original assignee: Freeline Therapeutics Limited
Priority date: 2021-11-04
Filing date: 2022-11-04
Publication date: 2023-05-11
Also published as: WO2023079301A1; TW202323270A

Abstract

The present invention relates to a modified complement factor I (CFI) polypeptide comprising one or more mutations, wherein the one or more mutations provides higher CFI activity. The present invention also relates to a polynucleotide comprising a CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes the modified CFI polypeptide of the invention. The present invention further relates to a viral particle comprising a recombinant genome comprising the polynucleotide of the invention; a composition comprising the polypeptide, polynucleotide or viral particle of the invention; and methods and uses of the polypeptide, polynucleotide, viral particle or composition of the invention.

Description

MUTANT COMPLEMENT FACTOR I VARIANTS WITH INCREASED ACTIVITY

Field of the invention

The present invention relates to a modified complement factor I (CFI) polypeptide and a polynucleotide comprising a modified CFI nucleotide sequence. The invention further relates to a viral particle comprising a recombinant genome comprising the polynucleotide of the invention, and a composition comprising the modified CFI polypeptide, polynucleotide, or viral particle of the invention. The invention also relates to methods of using, and uses of, the modified CFI polypeptide, polynucleotide, viral particle and/or composition of the invention. The invention further relates to the modified CFI polypeptide, polynucleotide, viral particle, or composition of the invention for use in a method of treatment, or use in the manufacture of a medicament for use in a method of treatment.

Background of the invention

Complement Factor I (CFI) is a protein involved in the complement system. The complement system is a part of the innate immune system, and forms a first line of defence against infections by triggering inflammatory responses. The complement system comprises a number of proteins that are synthesised primarily by the liver and circulate in the blood as inactive precursors. The stimulation of the complement system, for instance by a foreign entity, triggers proteases in the system to cleave the precursors and release molecules such as cytokines to initiate a further amplifying cascade. The activation of the complement system can stimulate phagocytes, stimulate inflammation and activate the cell-killing membrane attack complex. The complement system can be activated by the classical pathway, lectin pathway and alternative pathway. All three pathways converge at the cleavage and activation of C3 by C3-convertase. C3a and C3b are formed, causing a cascade of further cleavage and activation events. The alternative pathway is constitutively active at low levels (so called “tick-over”) and can also serve as an amplification loop when C3b is released into the fluid phase or, more typically, becomes opsonized to a foreign entity. Also, the alternative pathway can be triggered by damaged tissues or propagated on tissues lacking essential regulators. Under normal circumstances, the alternative pathway is governed by a balance between two competing cycles, namely a C3b feedback cycle that enhances complement amplification and a C3b breakdown cycle that leads to down regulation.

The alternative pathway is continuously activated at a low level due to spontaneous C3 hydrolysis to form C3b. C3b can be covalently bound to the surface of a cell or pathogen. The surface-bound C3b can combine with Factor B to form a complex, C3bB. C3bB can be cleaved in the presence of Factor D to form C3bBb (alternative pathway C3-convertase) that can convert more C3 into C3b. The complexed C3bBb can bind Factor P (properdin) to form the stabilised C3-convertase C3bBbP, which is able to cleave more C3 to C3b, thereby amplifying the response. When the C3bBbP complex forms on a pathogen or cell surface, it is able to bind an additional C3b to form alternative pathway C5-convertase, consisting of (C3b)2BbP. C5-convertase cleaves C5 into C5a and C5b. The membrane attack complex can then be formed from C5b binding sequentially to C6, C7, C8 and multiple C9 molecules.

A C3 breakdown cycle exists to prevent excessive C3b generation. In particular, CFI plays a role in the C3 breakdown cycle. Its role is to regulate complement activation by cleaving C3b and iC3b in two steps. In the first step, CFI, in the presence of the co-factor Factor H, catalyses the cleavage of C3b to iC3b. iC3b cannot form part of a C3 or C5 convertase, but is pro-inflammatory. In the second step, CFI, in the presence of the cofactor CR1 (CD35), then further breaks down iC3b yielding a C3 fragment, C3d,g.

The complement system plays a role in many diseases with an immune component.

Dysregulation of pathways in the complement system results in the downstream production of harmful inflammatory species that may lead to diseases of alternative (e.g. C3 glomerulopathy, dry AMD and geographic atrophy (GA)) or terminal (e.g. paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uremic syndrome (aHUS)) pathway dysregulation, or potentially any disease where complement mediated inflammation downstream of C3 is a contributing factor to disease pathogenesis (e.g. lupus nephritis, IgA nephropathy, and C3 glomerulopathy). Dysregulation of these pathways therefore has implications. For example, if CFI is not functioning or lacking, or the alternative pathway is over stimulated or inappropriately stimulated, the balance of the complement system can then be disrupted, leading to a build-up of inflammatory factors e.g. in the kidney, which can lead to kidney pathology. Thus, it is desirable to rebalance the cycles of the alternative pathway, particularly by promoting C3b and iC3b breakdown, and thereby reduce inflammation. Thus, it would be therapeutically advantageous to restore or increase the level of CFI, particularly in complement-mediated disorders.

Accordingly, there is a need to provide improved and effective therapy for the treatment of complement-mediated disorders which would allow the expression levels and/or activity levels of functional CFI to be restored or increased, to thereby effectively rebalance the cycles of the alternative pathway.

Summary of the invention

The present invention relates to a modified CFI polypeptide. The present Examples demonstrate that mutations at certain positions of a CFI polypeptide can be used to improve/increase the activity of the CFI polypeptide. Specifically, the Examples demonstrate that modified CFI polypeptides comprising mutations at positions corresponding to positions 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1, alone or in combination, demonstrate higher activity compared to corresponding CFI polypeptides that lack mutations at positions corresponding to positions 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. Such modified CFI polypeptides may be used in therapy to rebalance the cycles of the alternative pathway more effectively than a wild type CFI polypeptide. Furthermore, nucleotides encoding such modified CFI polypeptides may be used in gene therapy to effectively rebalance the cycles of the alternative pathway.

Accordingly, in a first aspect, the present invention provides a modified CFI polypeptide comprising one or more mutations, wherein the one or more mutations provides higher CFI activity.

In a second aspect, the present invention provides a modified CFI polypeptide comprising one or more mutations, wherein the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

In a third aspect, the present invention provides a polynucleotide comprising a CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes the modified CFI polypeptide of the invention.

In a fourth aspect, the present invention provides a viral particle comprising a recombinant genome comprising the polynucleotide of the invention.

In a fifth aspect, the present invention provides a composition comprising the modified CFI polypeptide, polynucleotide or viral particle of the invention, and a pharmaceutically acceptable excipient.

In a sixth aspect, the present invention provides the modified CFI polypeptide, polynucleotide, viral particle, or composition of the invention for use in a method of treatment.

In a seventh aspect, the present invention provides a method of treatment comprising administering an effective amount of the modified CFI polypeptide, polynucleotide, viral particle, or composition of the invention. In an eighth aspect, the present invention provides use of the modified CFI polypeptide, polynucleotide, viral particle, or composition of the invention in the manufacture of a medicament for use in a method of treatment.

Description of the figures

Figure 1 provides a comparison of the iC3b formation activity level of the various CFI variants comprising single amino acid mutations (listed in Table 2) and wild-type CFI (PL01). The activity levels are plotted as fold-changes relative to the activity level for wild-type CFI. (In particular, the HTRF ratio for each CFI variant has been divided by the HTRF ratio for wild-type CFI.) Error bars = mean ± SD. “100% iC3b” is a control showing the HTRF ratio (plotted as a fold-change relative to the HTRF ratio of wild-type CFI) achieved for 3nM of iC3b in the absence of any CFI. PL09 was included as a positive control.

Figure 2 provides a time-response curve for some of the highest CFI variants comprising single amino acid mutations. The iC3b formation rate of these single mutants are compared to wild-type CFI (WT). The “iC3b formation rate (% of 3nM iC3b)” shows for a particular reaction time the percentage of iC3b that has formed, where 100% represents the formation of 3nM iC3b. The “reaction time” corresponds to the total length of time the CFI variant or wild type CFI has been incubated with C3b and CFH and the antibodies. Error bars = mean ± SD.

Figure 3 provides a comparison of the iC3b formation level of the various CFI variants comprising double amino acid mutations (listed in Table 2) and wild-type CFI (PL01). The activity levels are plotted as fold-changes relative to the activity level for wild-type CFI. (In particular, the HTRF ratio for each CFI variant has been divided by the HTRF ratio for wild-type CFI.) Error bars = mean ± SD. “100% iC3b” is a control showing the HTRF ratio (plotted as a fold-change relative to the HTRF ratio of wildtype CFI) achieved for 3nM of iC3b in the absence of any CFI.

Figure 4 shows an outline of the steps of complement activation. FD, FB, FH, and FI stand for Factors D, B, H, and I respectively.

Figure 5 shows the feedback loop of the alternative pathway of vertebrate complement. Dashed lines indicate enzymatic reaction. Solid lines indicate conversions of proteins. D, B, H, and I stand for Factors D, B, H, and I respectively.

Figure 6 - Sequence Listing

Figures 7A and B show a comparison of the iC3b formation level of the various CFI variants comprising triple, quadruple or quintuple amino acid mutations (listed in Table 2), wild-type CFI (PL01) and the CFI variants PL120, PL133, PL135 and PL141 comprising double amino acid mutations. The activity levels are plotted as fold-changes relative to the activity level for wild-type CFI. (In particular, the HTRF ratio for each CFI variant has been divided by the HTRF ratio for wild-type CFI.) Error bars = mean ± SD. “100% iC3b” is a control showing the HTRF ratio (plotted as a fold-change relative to the HTRF ratio of wildtype CFI) achieved for 3nM of iC3b in the absence of any CFI.

Figure 8 shows the Wimley- White hydrophobicity scale for the free energy (AG) transition of an amino acid from an aqueous phase to a non-aqueous phase (octanol). A more negative AG value is associated with a more favourable transition from the aqueous phase into the non-aqueous phase, and denotes a more hydrophobic amino acid.

Description of the Sequence Listing

Detailed description

General definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “a modified CFI polypeptide comprising two mutations ” should be interpreted to mean that the modified CFI polypeptide has at least two mutations, but may contain further mutations. Similarly, the phrase “a polynucleotide comprising a CFI nucleotide sequence ” refers to a polynucleotide that has a CFI nucleotide sequence, but the polynucleotide may contain additional nucleotides.

In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting essentially of’ . The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of ’ . The term “consisting of” is intended to be limiting. For example, the phrase “a modified CFI polypeptide consisting of two mutations ” should be interpreted to mean that the modified CFI polypeptide has two mutations only and no additional mutations. Similarly, the phrase “a polynucleotide consisting of a CFI nucleotide sequence ” should be understood to mean that the polynucleotide has a CFI nucleotide sequence and no additional nucleotides.

In some embodiments of the invention, the word “have ” can be replaced with the word “comprise ”, the phrase “consist essentially of”, or the phrase “consist of”. In some embodiments of the invention, the word “has ” can be replaced with the word “comprises ”, the phrase “consists essentially of”, or the phrase “consists of”.

As used herein, “between” when referring to two endpoints to define a range of values should be taken to mean “between and including”. Thus, a range defined as “between 400 and 583” includes all values greater than 400 and less than 583, as well as the discrete values 400 and 583 themselves.

The terms “protein ” and “polypeptide ” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length. The terms “Factor I”, “CFI” and “Complement Factor I” are used interchangeably herein.

The terms “Factor H”, “cofactor H”, “co-factor H”, “CFH” and “Complement Factor H” are used interchangeably herein.

The terms “mutation” , “substitution mutation” and “amino acid substitution” are used interchangeably herein, and are intended to mean the substitution of one amino acid in an amino acid sequence with a different amino acid. For example, a modified CFI polypeptide comprising a mutation at a position corresponding to position 53 of SEQ ID NO: 1 may correspond to a CFI polypeptide that has the sequence of SEQ ID NO: 1 except that the amino acid at position 53 of the CFI polypeptide is different to the amino acid at position 53 in SEQ ID NO: 1. Alternatively, a modified CFI polypeptide comprising a mutation at a position corresponding to position 53 of SEQ ID NO: 1 may correspond to a CFI polypeptide that has the sequence of SEQ ID NO: 1, except that a few amino acids including the amino acid at position 53 are different to the corresponding amino acids in SEQ ID NO: 1 (for example a contiguous portion of the CFI polypeptide comprising position 53 may contain amino acids which are different from the corresponding amino acids in SEQ ID NO: 1). In the phrases “the mutation at a position corresponding to position X of SEQ ID NO: Z is a substitution with amino acid Y” and “the one or more mutations comprises a mutation at a position corresponding to position X of SEQ ID NO: Z”, the substituted residue is the amino acid at the position corresponding to position X of SEQ ID NO: Z. Amino acid Y is the different amino acid which replaces the original or native amino acid in an amino acid sequence at the position corresponding to position X of SEQ ID NO: Z. In the phrases “substitution of amino acid X or “amino acid X that is (to be) substituted’' , amino acid X is the original or native amino acid that is present within an amino acid sequence and that is to be replaced. For example, substitution of arginine means that an original or native arginine amino acid is replaced by another amino acid. In the phrase “substitution with” amino acid Y or “mutation to ” amino acid Y, amino acid Y is the different amino acid which replaces the original or native amino acid in an amino acid sequence. For example, substitution with leucine refers to replacement of an original or native (non-leucine) amino acid with leucine. The standard shorthand nomenclature used to define a substitution mutation lists the original or native amino acid at a position within an amino acid sequence that is to be substituted, and the amino acid which replaces the original or native amino acid. For example, a modified CFI polypeptide comprising the substitution mutation R53L refers to a modified CFI amino acid sequence which comprises a substitution of the arginine residue at a position corresponding to position 53 with a leucine residue (z.e. which comprises a leucine residue at a position corresponding to position 53).

Amino acids “corresponding to ” specified positions of a specified SEQ ID NO may be amino acids at the specified positions of the particular SEQ ID NO recited. For example, the amino acid “corresponding to position 53 of SEQ ID NO: 1 ” may be the amino acid at position 53 of SEQ ID NO: 1. Alternatively, amino acids “corresponding to ” specified positions of a specified SEQ ID NO may be amino acids from an alternative amino acid sequence which correspond to the specified positions of the specified SEQ ID NO. For example, the amino acid “corresponding to position 53 of SEQ ID NO: 1 ” may be the amino acid from an alternative amino acid sequence which corresponds to position 53 of SEQ ID NO: 1. It is within the capabilities of the person skilled in the art to determine which amino acids in an alternative amino acid sequence “correspond to ” the specified positions in the specified SEQ ID NO. For example, the person skilled in the art merely needs to perform a sequence alignment of the alternative amino acid sequence with the specified SEQ ID NO using a suitable alignment algorithm such as that of Needleman and Wunsch described herein, and determine which region of the alternative amino acid sequence aligns to the specified positions in the specified SEQ ID NO. For example, the skilled person is able to align the alternative amino acid sequence with SEQ ID NO: 1 and determine which amino acid aligns, and therefore corresponds to, e.g. position 53 of SEQ ID NO: 1. The terms “wild-type ” and “native ” are used interchangeably herein, and are intended to describe something which is naturally occurring. For example, a “wild-type CFI amino acid sequence ” is a CFI amino acid sequence which occurs in nature.

The terms “AA V viral particle ” and “AA V vector ” are used interchangeably herein.

The term “around” used in the context of describing the length of nucleotide or amino acid sequences indicates that a sequence may comprise or consist of a defined number of nucleotides or amino acids, plus or minus 10%, more particularly plus or minus 5%, more particularly plus or minus 1%, or more particularly plus or minus a single integer. For example, reference to a nucleotide sequence of “around” 1698 nucleotides in length may refer to a nucleotide sequence of 1529-1867 nucleotides, more particularly 1614-1782 nucleotides, more particularly 1682-1714 nucleotides, and more particularly 1697-1699 nucleotides in length.

The term “around” used in the context of a length of time (e.g. around 16 hours) indicates that the length of time includes the specified length of time plus or minus 10%, more particularly plus or minus 5%, or more particularly plus or minus 1%.

For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid at the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions /total number of positions in the reference sequence x 100). Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 1. If the test sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.

Similarly, to determine whether a “test” sequence is or comprises a sequence that is at least 95% identical to a fragment of SEQ ID NO: 1, the skilled person would align the test sequence with SEQ ID NO: 1 and identify a contiguous portion of the reference sequence of the required length which best aligns with the test sequence ( “reference fragment”). The corresponding portion of the “test” sequence which aligns to the “reference fragment” is the “test fragment”. The skilled person would then calculate the percentage identity between and “test fragment” and the “reference fragment”, using the calculation % identity = (number of positions that are identical between the “test fragment” and the “reference fragment” /the length of the “reference fragment”) x 100. For example, to determine whether a “test” sequence is or comprises a sequence that is at least 95% identical to a fragment of at least 400 amino acids of SEQ ID NO: 1, the skilled person would align the test sequence with SEQ ID NO: 1, and identify a contiguous 400 amino acid portion of SEQ ID NO: 1 which best aligns with the test sequence (in this example, this would be the “reference fragment”). The corresponding portion of the “test” sequence which aligns to the 400 amino acid portion of SEQ ID NO: 1 is the “test fragment” in this example. The user then calculates the percentage identity between the “test fragment” and the 400 amino acid portion of SEQ ID NO: 1 that aligns to the test fragment (“reference fragment”) as described above, i.e. using the calculation % identity = number of positions that are identical between the “test fragment” and the “reference fragment” 7400 (the length of the “reference fragment”) x 100.

The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, alignments may be performed using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The singular forms “a”, “an ”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an amino acid” includes two or more instances or versions of such amino acids.

For the purposes of the present invention, the term “fragment” refers to a contiguous portion of a sequence. For example, a fragment of SEQ ID NO: 1 of 50 amino acids refers to 50 contiguous amino acids of SEQ ID NO: 1.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

A modified CFI polypeptide

The present invention provides a modified CFI polypeptide (also referred to as a CFI variant) comprising one or more mutations. The term “modified” means that the polypeptide has at least one difference compared to a wild-type CFI polypeptide, e.g. a mutation has been introduced. Without wishing to be bound by theory, it is believed that the instant amino acid mutations may serve to enhance the binding affinity between CFI and its substrates and cofactors, thereby providing higher CFI activity (e.g. catalysing the cleavage of C3b to iC3b, and/or degrading iC3b). For example, this may be achieved by substituting an amino acid in CFI that is located at the interface between CFI and its substrate C3b or at the interface between CFI and its cofactor, Factor H.

Optionally, the one or more mutations comprises (or consists of) 20 or fewer, 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 10 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 5 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 4 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 3 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) 2 or fewer mutations. Optionally, the one or more mutations comprises (or consists of) one mutation.

CFI polypeptide is a glycoprotein heterodimer consisting of a heavy chain and light chain. The heavy chain consists of an FI membrane attack complex domain (FIM domain), a CD5 domain, a low-density lipoprotein receptor 1 domain, a low-density lipoprotein receptor 2 domain and the D-region (region of no known homology). The light chain consists of the serine protease domain (SP domain). The SP domain comprises the residues that form the catalytic triad. C3b is a substrate of CFI and comprises an a’ -chain and a P chain. The a’- chain comprises the C-terminal C345c (CTC) domain, the ‘complement Clr/Cls, UEGF, BMP1’ (CUB) domain, and the thio-ester-containing (TED) domain. The CUB domain comprises the substrate loop. CFI cleaves C3b to iC3b by mediating two cuts in the a’- chain of C3b. A small fragment (C3f; 3kDa) is released and the a’-chain is divided into a 68kDa fragment and a 43kDa fragment. The proteolysed C3b is called iC3b. Factor H is a cofactor involved in the catalysis of the cleavage of C3b into iC3b. Factor H consists of 20 complement control protein (CCP) domains, including CCP2 and CCP3. The skilled person is aware of the location of the various domains based on known crystal structures of CFFC3b/F actor H (for example, PDB: 5032).

CFI plays a role in the C3 breakdown cycle. CFI can regulate complement activation by inactivating C3b and degrading iC3b. CFI can catalyse the cleavage of C3b to iC3b (also referred to as “C3bdnactivating” activity or “iC3b formation” activity). CFI can breakdown iC3b to C3d,g (also referred to as “iC3b-degradation ” activity). A typical wild type CFI polypeptide is encoded by SEQ ID NO: 3 or 4. Optionally, a wild type CFI polypeptide is encoded by SEQ ID NO: 28. Optionally, a wild type CFI polypeptide is encoded by SEQ ID NO: 19.

Typically, CFI polypeptide is initially expressed as a precursor “immature” form (e.g. a CFI of SEQ ID NO: 1 encoded by SEQ ID NO: 3 or SEQ ID NO: 28), comprising a signal peptide (e.g. amino acid residues 1 to 18 of SEQ ID NO: 1 and codons 1 to 18 of SEQ ID NO: 3 or SEQ ID NO: 16), and a mature CFI polypeptide region. After processing, the “mature” form of CFI lacks the signal peptide. The term “mature CFI” or “mature CFI polypeptide” refers to a CFI polypeptide that does not comprise the signal peptide, such as a CFI encoded by SEQ ID NO: 4. The CFI polypeptide sequence of SEQ ID NO: 2 is a mature CFI. A typical wild type CFI signal peptide may be encoded by a nucleotide sequence of SEQ ID NO: 5 and have the polypeptide sequence of SEQ ID NO: 6.

Optionally, the modified CFI polypeptide of the invention comprises a signal peptide. Optionally, the amino acid sequence of the signal peptide is an amino acid sequence of a wild type (native) CFI signal peptide. The amino acid sequence of a wild type CFI signal peptide may be the amino acid sequence of SEQ ID NO: 6. Optionally, the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide. For example, the signal peptide may not be a wild type CFI signal peptide. The signal peptide may be the signal peptide from another polypeptide. The signal peptide may be a heterologous signal peptide. The heterologous signal peptide may be, for example, a signal peptide from another polypeptide, a modified CFI signal peptide, or a synthetic signal peptide. In such embodiments, a “modified CFI signal peptide ” is a wild type CFI signal peptide which has been modified (e.g. a mutation has been introduced).

Optionally, the modified CFI polypeptide of the invention does not comprise a signal peptide.

The modified CFI polypeptide of the invention is functional. A functional CFI polypeptide is one which can regulate complement activation by inactivating C3b (i.e. forming iC3b) and degrading iC3b as described herein. It is within the abilities of the person skilled in the art to determine whether a CFI polypeptide or a fragment thereof is functional. The skilled person merely needs to test whether the polypeptide is active. If the modified CFI polypeptide has at least 20% (optionally, at least 30%, at least 40%, at least 50%, or at least 75%) of the activity of a wild-type CFI polypeptide (determined using the same method), then it is functional.

Preferably, the modified CFI polypeptide of the invention is a modified human CFI polypeptide.

In an embodiment, the modified CFI polypeptide has the same or higher activity than a wild-type CFI polypeptide, such as a wild-type CFI polypeptide of SEQ ID NO: 1. For example, the modified CFI polypeptide may have an activity at least 1.1 -fold, at least 1.2- fold, or at least 1.5-fold higher than the wild-type CFI polypeptide of SEQ ID NO: 1.

In an embodiment, the modified CFI polypeptide has the same or higher activity than the wild-type CFI polypeptide of SEQ ID NO: 2. For example, the modified CFI polypeptide may have an activity at least 1.1 -fold, at least 1.2-fold, or at least 1.5-fold higher than the wild-type CFI polypeptide of SEQ ID NO: 2. In some embodiments, the one or more mutation provides higher CFI activity. Exemplary mutations which provide higher CFI activity are disclosed herein. By specifying that the “one or more mutations provides higher CFI activity” , the modified CFI polypeptide comprising the one or more mutations has higher CFI activity relative to a reference CFI polypeptide that does not comprise the one or more mutations but is otherwise identical to the modified CFI polypeptide comprising the one or more mutations. For example, if the one or more mutations provides higher CFI activity and the modified CFI polypeptide comprising the one or more mutations is otherwise identical to a wild type CFI polypeptide except that it comprises the one or more mutations (e.g. mutations R53L and R383Y), the modified CFI polypeptide comprising the one or more mutations has higher CFI activity than the wild type CFI polypeptide. In some embodiments, the one or more mutations “provides higher CFI activity” if a “test” polypeptide that is identical to SEQ ID NO: 1 except for the one or more mutations has higher CFI activity compared to the polypeptide of SEQ ID NO: 1, optionally where the “test” polypeptide and SEQ ID NO: 1 are produced by expression in the same cell line. The modified CFI polypeptide comprising the one or more mutations may not necessarily have higher CFI activity than a wild type CFI polypeptide. For example, the modified CFI polypeptide comprising the one or more mutations may comprise one or more further modifications (such as a deleted portion) which reduce the CFI activity of the polypeptide. In such a case, the one or more mutations disclosed herein may serve to provide higher activity for the modified CFI polypeptide such that the modified CFI polypeptide may not have the same CFI activity as a wild type CFI polypeptide but nevertheless has higher CFI activity than a reference CFI polypeptide that is otherwise identical to the modified CFI polypeptide but lacks the one or more mutations. Alternatively, the one or more mutations may provide increased CFI activity which restores the CFI activity of the modified CFI polypeptide to the level of CFI activity of a wild type CFI polypeptide.

In some embodiments, the modified CFI polypeptide comprising the one or more mutations has higher CFI activity. In some embodiments, the modified CFI polypeptide comprising the one or more mutations has higher CFI activity relative to a reference CFI polypeptide as described herein. For example, the reference CFI polypeptide may be a wild-type CFI polypeptide. The reference CFI polypeptide may be the polypeptide of SEQ ID NO: 1. The reference CFI polypeptide may be the polypeptide of SEQ ID NO: 2. In some embodiments, the CFI activity of a modified CFI is compared to the CFI activity of a reference CFI polypeptide. Exemplary mutations which increase the CFI activity of a modified CFI polypeptide are disclosed herein.

The “reference CFI polypeptide” is a CFI polypeptide that does not comprise the one or more mutations of the modified CFI polypeptide of the invention. Optionally, the reference CFI polypeptide does not comprise the one or more mutations but is otherwise identical to the modified CFI polypeptide of the invention. The reference CFI polypeptide may be a wild-type CFI polypeptide. The reference CFI polypeptide may be the polypeptide of SEQ ID NO: 1 or 2. Optionally, the reference CFI polypeptide is a CFI polypeptide that has the same sequence as SEQ ID NO: 1 or 2, except that it comprises a mutation to valine at a position corresponding to position 138 of SEQ ID NO: 1, optionally M138V.

For the purposes of determining whether a modified CFI polypeptide has higher CFI activity relative to a reference CFI polypeptide (as described herein, such as a wild-type CFI polypeptide), the CFI activity of the modified CFI polypeptide and reference CFI polypeptide are determined using the same method and the results compared. The method may be any of the methods described herein for determining CFI activity.

Optionally, the modified CFI polypeptide has a CFI activity which is at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, between 1.2 and 3 fold, between 1.2 and 5 fold, between 2 fold and 6 fold, between 3 fold and 8 fold, between 1.5 fold and 8 fold, between 2 fold and 8 fold, between 5 fold and 8 fold, between 6 fold and 8 fold, between 7 fold and 8 fold, between 2 fold and 15 fold, between 3 fold and 20 fold, between 5 fold and 10 fold, between 5 fold and 15 fold, or between 5 fold and 20 fold higher than the CFI activity of the reference CFI polypeptide (such as a wild-type CFI polypeptide). When referring to fold changes of activity, the term “between ” includes the specified values. Thus, for example, “between 1.5 fold and 8 fold” includes the values 1.5 fold and 8 fold.

Optionally, the modified CFI polypeptide has a CFI activity which is at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, or at least 6 fold higher than the CFI activity of the reference CFI polypeptide (such as a wild-type CFI polypeptide). Optionally, the modified CFI polypeptide has a CFI activity which is 20 fold or less, 15 fold or less, or 10 fold or less than the CFI activity of the reference CFI polypeptide (such as a wild-type CFI polypeptide). Optionally, the modified CFI polypeptide has a CFI activity which is between 1.2 fold and 20 fold, between 1.2 fold and 15 fold, between 1.5 fold and 15 fold, or between 1.5 and 10 fold higher than the CFI activity of the reference CFI polypeptide (such as a wild-type CFI polypeptide).

Optionally, the modified CFI polypeptide has a CFI activity which is between 1.2 fold and 6 fold or between 5 and 10 fold higher than the CFI activity of the reference CFI polypeptide (such as a wild-type CFI polypeptide).

Optionally, the CFI activity of the modified CFI polypeptide is iC3b formation activity. Optionally, the CFI activity of the modified CFI polypeptide is determined using a homogeneous time resolved fluorescence (HTRF) assay. Optionally, the CFI activity of the modified CFI polypeptide is measured after 1 hour of incubation at pH 7 with C3b and CFH, followed by 1 hour of incubation with an anti-iC3b IgG and an anti-C3b IgG.

Optionally, the CFI activity is measured after 30 minutes of incubation at pH 7 with C3b and CFH, followed by 30 minutes of incubation with an anti-iC3b IgG and an anti-C3b IgG. Optionally, the anti-iC3b IgG is an anti-iC3b (Tb) IgG and the anti-C3b IgG is an anti-C3b (FITC) IgG. Activity (also referred to as “CFI activity”) of a CFI polypeptide herein may refer to the observed activity in a functional assay for determining the activity of the CFI polypeptide. The activity may be the specific activity which refers to the activity per unit of CFI polypeptide such that the activity is normalised to take account of the amount or concentration of CFI polypeptide in the sample. This can be done by measuring the concentration of the CFI polypeptide in the sample, for example by using a standard ELISA assay, and dividing the activity by the CFI concentration. The activity of the CFI polypeptide can be evaluated by measuring CFI mediated-cleavage of C3b into iC3b (also referred to as “CFI iC3b formation activity”, “iC3b formation activity”, or “C3b degradation activity”) using antibodies specific for an iC3b neoepitope, for example using a homogenous time resolved fluorescence (HTRF) assay, such as those described herein. For the purposes of the present application, the terms “iC3b formation activity”, “C3b- inactivating activity” and “C3b-degradation activity” should be considered to be synonyms. For example, a suitable assay is disclosed in Example 1 under the heading “ CFI activity determination through measurement of iC3b formation using an HTRF assay”. Suitable methods for evaluating the activity of the CFI polypeptide are discussed below.

The iC3b-formation activity of a CFI polypeptide can be assessed by a person skilled in the art. For example, a sample comprising the polypeptide to be tested is mixed with Factor H and C3b. The concentration of the polypeptide in the sample to be mixed with Factor H and C3b could be 150 pM. The sample could be mixed with Factor H and C3b so that the concentration of polypeptide is 75 pM, the concentration of C3b is 4.5 nM and the concentration of Factor H is 3 nM. The mixture could be incubated for 1 hour. The incubation could be at room temperature. Cleavage of C3b to iC3b could then be measured using an antibody that only binds a human iC3b neoantigen (i.e. an antibody which only binds iC3b and not C3b) and an antibody that binds to both C3b and iC3b. The antibody that only binds a human iC3b neoantigen could be a monoclonal IgG antibody conjugated to terbium cryptate (e.g. from CisBio, such as the antibody specified in Example 1). The antibody that binds to both C3b and iC3b could be conjugated to fluorescein (FITC) (e.g. from Cedarlane, such as the antibody specified in Example 1). Both antibodies could be added to the mixture of the polypeptide, Factor H and C3b so that the concentration of the polypeptide is 50 pM, the concentration of C3b is 3 nM and the concentration of Factor H is 2 nM. The concentration in the mixture of the antibody that only binds a human iC3b neoantigen could be 0.5 pg/mL and the concentration of the antibody that binds to both C3b and iC3b could be 12 nM. The mixture could be incubated for 1 hour further. The iC3b-formation activity could then be assessed fluorometrically as discussed below.

To assess the iC3b-formation activity fluorometrically, the mixture to be tested (e.g. the mixture following the incubation with the antibodies) could be exposed to an excitation wavelength of 340 nm (e.g. using a SpectraMax® i3 plate reader; Molecular Devices). Fluorescent readings could then be taken. The fluorescent readings could be taken at 490 nM for the emission spectra of terbium cryptate and at 520 nm for the emission spectra of FITC. The mean fluorescence intensity (MFI) could then be calculated for both the 490 nm and 520 nm readings. If iC3b has been formed, then both antibodies will bind iC3b and the terbium cryptate and fluorescein will be brought close enough together for FRET to occur. The level of FRET (as measured by MFI at 490nm and 520nm) will be proportional to the amount of iC3b produced. The HTRF ratio could then be calculated by dividing the MFI measured at 520nm (HTRF acceptor) by the MFI measured at 490 nm (HTRF donor), and multiplying the resulting value by 10,000.

A further example of how the iC3b-formation activity of a CFI polypeptide can be assessed by a person skilled in the art is as follows. For example, a sample comprising the polypeptide to be tested is mixed with Factor H and C3b. The concentration of the polypeptide in the sample to be mixed with Factor H and C3b could be 75 pM. The sample could be mixed with Factor H and C3b so that the concentration of polypeptide is 37.5 pM, the concentration of C3b is 4.5 nM and the concentration of Factor H is 3 nM. The mixture could be incubated for 30 minutes. The incubation could be at room temperature. Cleavage of C3b to iC3b could then be measured using an antibody that only binds a human iC3b neoantigen (i.e. an antibody which only binds iC3b and not C3b) and an antibody that binds to both C3b and iC3b. The antibody that only binds a human iC3b neoantigen could be a monoclonal IgG antibody conjugated to terbium cryptate (e.g. from CisBio, such as the antibody specified in Example 1). The antibody that binds to both C3b and iC3b could be conjugated to fluorescein (FITC) (e.g. from Cedarlane, such as the antibody specified in Example 1). Both antibodies could be added to the mixture of the polypeptide, Factor H and C3b so that the concentration of the polypeptide is 25 pM, the concentration of C3b is 3 nM and the concentration of Factor H is 2 nM. The concentration in the mixture of the antibody that only binds a human iC3b neoantigen could be 0.5 pg/mL and the concentration of the antibody that binds to both C3b and iC3b could be 12 nM. The mixture could be incubated for 30 minutes further. The iC3b- formation activity could then be assessed fluorometrically as discussed above.

The concentration of the polypeptide can be determined using a suitable assay, such as an ELISA. Suitable ELISA assays are described herein. An ELISA such as that described in Example 1 could be used.

The polypeptide to be tested may be comprised in a supernatant. Thus, the sample, comprising the CFI polypeptide, may be a supernatant. The polypeptide to be tested may have been expressed and secreted into a supernatant following the transfection of cells with a polynucleotide comprising a nucleotide sequence which encodes for the polypeptide. A suitable method for obtaining a supernatant comprising the polypeptide to be tested is as follows. A polynucleotide comprising a nucleotide sequence which encodes the polypeptide (e.g. a transgene-containing plasmid as described in Example 1) can be transfected into cells (e.g. Expi293F cells), for example, by using a transfection reagent. Optionally, a polynucleotide comprising a nucleotide sequence which encodes Furin (e.g. human Furin) can also be transfected into the cells. The cells can then be incubated to allow expression and secretion of the polypeptide into the supernatant. The supernatant can then be collected. The supernatants comprising the polypeptide may be obtained according to the protocol described in Example 1. Optionally, when testing the polypeptide, the polypeptide is tagged with a His-tag (e.g. a 6His-tag). For example, when being tested, the polypeptide may comprise a His-tag (e.g. a 6His-tag) at the N-terminus of the mature CFI polypeptide. If the polypeptide to be tested is expressed and secreted into a supernatant following the transfection of cells with a polynucleotide comprising a nucleotide sequence which encodes for the polypeptide, the nucleotide sequence may encode a His-tag (e.g. a 6His-tag) at the N-terminus of the sequence encoding the mature CFI polypeptide. For example, the nucleotide sequence may encode a His-tag (e.g. a 6His- tag) between a sequence encoding the signal peptide and a sequence encoding the mature CFI polypeptide. For example, a transgene may be prepared comprising a sequence encoding a signal peptide, a sequence encoding a 6His-tag and a sequence encoding a mature CFI sequence as set out in Example 1. As an example, if the polypeptide to be tested comprises a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, then the transgene may comprise the sequence of SEQ ID NO: 17.

In an example of an ELISA assay, an antibody that binds to the CFI polypeptide could be bound to a plate. The sample, comprising the CFI polypeptide at unknown concentration, could be passed over the plate. A second detection antibody that binds to the CFI polypeptide could be applied to the plate, and any excess washed off. The detection antibody that remains (i.e. is not washed off) will be bound to the CFI polypeptide. The detection antibody could be linked to an enzyme such as horse radish peroxidase, or the detection antibody could be biotinylated. The level of detection antibody that binds to the CFI polypeptide on the plate could be measured by measuring the amount of the detection antibody. For example, if the detection antibody is linked to horse radish peroxidase, the horse radish peroxidase can catalyse the production of a blue reaction product from a substrate such as TMB (3,3’,5,5’-tetramethylbenzidine), and the level of the blue product can be detected by absorbance at 450 nm. As a further example, if the detection antibody is biotinylated, a streptavidin-peroxidase conjugate can be added in order to bind to the biotinylated detection antibody, and the peroxidase can catalyse the production of a blue reaction product from a substrate such as TMB (3,3’,5,5’-tetramethylbenzidine), and the level of the blue product can be detected by absorbance at 450 nm. The level of the blue product is proportional to the amount of detection antibody that remained after the washing step, which is proportional to the amount of the CFI polypeptide in the sample. The higher the level of the blue product, the higher the concentration of CFI polypeptide in the sample. The concentration of CFI polypeptide can be evaluated using a standard curve. For example, a standard curve can be obtained by obtaining the absorbances for a series of CFI standards of known concentrations (in addition to the sample) and plotting the absorbance (linear) of each standard against the corresponding concentration (log). The concentration of CFI polypeptide in the sample can then be determined from the standard curve. Alternatively, for example when using purified protein, the amount or concentration of CFI polypeptide may be determined spectrophotometrically.

Optionally, the one or more mutations comprises a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b. Optionally, the one or more mutations comprises a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b, and/or (ii) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H.

The phrase “the one or more mutations comprises a mutation at one or more positions within the modified CFI polypeptide which can form an interface with [polypeptide X] ” means that the mutation of the one or more mutations is at one or more positions within the modified CFI polypeptide that is/are able to form an interface with polypeptide X (which is C3b or Factor H). The mutation at one or more positions only needs to be able to form an interface with polypeptide X. It does not need to have formed an interface with polypeptide X. For example, the mutation at one or more positions may not be interacting with polypeptide X. Optionally, the modified CFI polypeptide may not be bound to polypeptide X. Alternatively, the mutation at one or more positions may be interacting with polypeptide X. Optionally, the modified CFI polypeptide may be bound to polypeptide X. Furthermore, where there are two or more mutations at an interface, the two or more mutations need not be immediately adjacent to one another in the primary, secondary or tertiary structure of the polypeptide. Rather, all that is required is that they both form part of the interface.

The “one or more positions within the modified CFI polypeptide which can form an interface with [polypeptide X] ” is/are considered to be any of the amino acid position(s) within the modified CFI polypeptide which correspond to the amino acid position(s) within a wild type CFI polypeptide (such as the polypeptide of SEQ ID NO: 1) that are within 6 Angstroms of an amino acid in polypeptide X when the wild type CFI polypeptide is interacting with or bound to polypeptide X. Polypeptide X is C3b or Factor H. A skilled person can readily determine whether two amino acids are within 6 Angstroms of each other by, for example, analysing the crystal structure. For example, it is possible to measure distances using any structural analysis software known in the art. Any other suitable methods known in the art may also be used by the skilled person to determine positions in a CFI polypeptide which can form an interface with polypeptide X, such as C3b or Factor H.

Optionally, the one or more mutations comprises a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b. Optionally, the one or more mutations comprises a mutation at two or more positions within the modified CFI polypeptide which can form an interface with C3b.

Optionally, the one or more mutations comprises a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises a mutation at two or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises a mutation at three or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises a mutation at four or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises a mutation at five or more positions within the modified CFI polypeptide which can form an interface with Factor H.

Optionally, the one or more mutations comprises (i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at two or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at three or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at four or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at two or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at two or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at two or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more mutations comprises (i) a mutation at two or more positions within the modified CFI polypeptide which can form an interface with C3b, and (ii) a mutation at three or more positions within the modified CFI polypeptide which can form an interface with Factor H. Optionally, the one or more positions within the modified CFI polypeptide which can form an interface with C3b is/are any of the amino acid position(s) within the modified CFI polypeptide which correspond to the amino acid position(s) selected from 53, 54, 55, 56, 58, 60, 63, 64, 65, 66, 69, 80, 81, 82, 83, 84, 87, 88, 90, 91, 94, 95, 360, 361, 362, 363,

364, 365, 379, 381, 383, 384, 385, 386, 389, 390, 414, 416, 417, 418, 419, 421, 422, 423,

424, 425, 426, 473, 474, 477, 479, 481, 499, 502, 511, 519, 520, 521, 522, 523, 524, 526,

544, 545, 546, 547, 548, 549, 550, 551, 552, 555, 556, 557, 558, 559, 567, 568, 570, 571,

572, 574, and 575 of SEQ ID NO: 1.

Optionally, the positions within the modified CFI polypeptide which can form an interface with C3b do not include any of the amino acid position(s) within the modified CFI polypeptide which correspond to the amino acid position(s) selected from 380, 429 and 525 of SEQ ID NO: 1. Optionally, the positions within the modified CFI polypeptide which can form an interface with C3b do not include any of the amino acid position(s) within the modified CFI polypeptide which correspond to the amino acid position(s) which form the active site. The active site of a wild type CFI polypeptide comprises a so-called ‘catalytic triad’ of amino acids which correspond to the amino acids at positions 380, 429 and 525 of SEQ ID NO: 1. Substituting one of these amino acids, or an amino acid in the vicinity of one of these amino acids, may reduce the catalytic activity of CFI.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain. Optionally, the positions within the modified CFI polypeptide in the SP domain which can form an interface with C3b is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 360, 361, 362, 363, 364, 365, 379, 381, 383, 384, 385, 386, 389,

390, 414, 416, 417, 418, 419, 421, 422, 423, 424, 425, 426, 473, 474, 477, 479, 481, 499,

502, 511, 519, 520, 521, 522, 523, 524, 526, 544, 545, 546, 547, 548, 549, 550, 551, 552,

555, 556, 557, 558, 559, 567, 568, 570, 571, 572, 574, and 575 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain and can form an interface with the CTC domain in the C3b alpha chain. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CTC domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 414, 416, 417, 418, 419, 567, 568, 570, 571, 572, 574, and 575 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CTC domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 419, 568, 571, 572, and 575 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain and can form an interface with the CUB domain in the C3b alpha chain. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CUB domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 361, 362, 363, 364, 365, 379, 381, 383, 384, 385, 386, 390, 417, 421, 422, 423, 424, 425, 426, 473, 474, 477, 479, 481, 499, 502, 511, 519, 520, 521, 522, 523, 524, 526, 544, 545, 546, 547, 548, 549, 550, 551, 552, 555, 556, 557, 558, and 559 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CUB domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 362, 384, 385, 386, 421, 423, 424, 426, 474, 479, 481, 502, 519, 520, 522, 523, 544, 545, 547, 548, 549, 555, and 557 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain and can form an interface with the substrate loop within the CUB domain in the C3b alpha chain. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the substrate loop within the CUB domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 361, 362, 363, 364, 365, 381, 423, 424, 426, 473, 474, 477, 479, 481, 499, 502, 511, 519, 520, 521, 522, 523, 524, 526, 544, 545, 546, 547, 548, 549, 550, 551, 552, 555, 556, 557, 558, and 559 of SEQ ID NO: 1. Optionally, the one or more positions within the modified CFI polypeptide in the SP domain which can form an interface with the substrate loop within the CUB domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 362, 423, 424, 426, 474, 479, 481, 502, 519, 520, 522, 523, 544, 545, 547, 548, 549, 555, and 557 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain and can form an interface with the C3b beta chain of C3b. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the C3b beta chain of C3b is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 360, 386, and 389 of SEQ ID NO: 1. Optionally, the one or more positions within the modified CFI polypeptide in the SP domain which can form an interface with the C3b beta chain of C3b is the position within the modified CFI polypeptide which corresponds to the position 386 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the FIM domain. Optionally, the positions within the modified CFI polypeptide in the FIM domain which can form an interface with C3b is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 53, 54, 55, 56, 58, 60, 63, 64, 65, 66, 69, 80, 81, 82, 83, 84, 87, 88, 90, 91, 94, and 95 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the FIM domain and can form an interface with the CTC domain in the C3b alpha chain. Optionally, at least one of the positions within the modified CFI polypeptide in the FIM domain which can form an interface with the CTC domain in the C3b alpha chain is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 53, 54, 55, 56, 58, 60, 63, 64, 65, 66, 69, 80, 81, 82, 83, 84, 87, 88, 90, 91, 94, and 95 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with Factor H is in the SP domain. Optionally, the positions within the modified CFI polypeptide in the SP domain which can form an interface with Factor H is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 345, 358, 360, 361, 362, 363, 364, 391, 393, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 436, 437, 438, 439, 440, 441, 442, 474, 478, 479, 480, 481, 482, and 483 of SEQ ID NO: 1.

Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with Factor H is in the SP domain and can form an interface with the CCP2 domain in Factor H. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CCP2 domain of Factor H is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 358, 360, 391, 393, 399, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 436, 437, 438, 439, 440, 441, and 442 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide in the SP domain which can form an interface with the CCP2 domain of Factor H is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 358, 391, 393, 399, 402, 404, 405, 406, 407, 408, 409, 410, 411, 437, 440, and 441 of SEQ ID NO: 1. Optionally, at least one of the positions within the modified CFI polypeptide which can form an interface with Factor H is in the SP domain and can form an interface with the CCP3 domain in Factor H. Optionally, the one or more positions within the modified CFI polypeptide in the SP domain which can form an interface with the CCP3 domain of Factor H is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 345, 358, 361, 362, 363, 364, 393, 399, 400, 401, 402, 404, 474, 478, 479, 480, 481, 482, and 483 of SEQ ID NO: 1. Optionally, the one or more positions within the modified CFI polypeptide in the SP domain which can form an interface with the CCP3 domain of Factor H is/are any of the position(s) within the modified CFI polypeptide which correspond to the position(s) selected from 358, 362, 393, 399, 402, 404, 474, 479, 481, and 482 of SEQ ID NO: 1.

The modified CFI polypeptide of the invention comprises one or more mutations (i.e. at least one substitution mutation). The one or more mutations (such as a mutation at a position corresponding to a position listed below) may provide one or more of the properties as discussed herein (e.g. higher CFI activity). The modified CFI polypeptide comprising one or more mutations (such as a mutation at a position corresponding to a position listed below) may have one or more of the properties as discussed herein (e.g. higher CFI activity).

In some embodiments, the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

Optionally, the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 401, 407, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 60, 383, 384, 401, 407, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1.

Optionally, the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 383, 401, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1.

Optionally, the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, 441, and 479 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, 441, and 479 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more or two or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, 441, and 479 of SEQ ID NO: 1.

Optionally, the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, and 479 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, and 479 of SEQ ID NO: 1. Optionally, the one or more mutations comprises a mutation at two or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, and 479 of SEQ ID NO: 1. Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation to a more hydrophobic amino acid. Biophysical studies have been performed to establish the hydrophobicity of the twenty naturally occurring amino acids, or more particularly, the relative hydrophobicity of the amino acids, and hydrophobicity scales list the hydropathy of each of the amino acids. One such example is the Wimley- White whole-residue hydrophobicity scale, which calculates the free energy of transfer of an amino acid from an aqueous phase to a non-aqueous phase (octanol). The term “a more hydrophobic amino acid” refers to an amino acid which has a more favourable (more negative AG) free energy value for the transition from an aqueous phase to octanol according to the Wimley- White hydrophobicity scale. The free energy for the aqueous phase to octanol transition is shown in Table 3 below, and is shown graphically in Figure 8. Starting with the most hydrophobic amino acid, the Wimley- White hydrophobicity scale lists the hydrophobicity of amino acids in the following order: tryptophan, phenylalanine, leucine, isoleucine, tyrosine, methionine, valine, cysteine, glutamic acid (uncharged), histidine (uncharged), proline, threonine, aspartic acid (uncharged), serine, alanine, glutamine, asparagine, glycine, arginine (positively charged), histidine (positively charged), lysine (positively charged), glutamic acid (negatively charged), aspartic acid (negatively charged).

Table 3 - Wimley- White whole residue hydrophobicity scale for water to octanol transition.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine or glutamic acid. Optionally, the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R53I. Optionally, the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L. Optionally, the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y. Optionally, the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally R53E.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 60 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 60 of SEQ ID NO: 1 is a substitution with serine or glutamic acid. Optionally, the mutation at a position corresponding to position 60 of SEQ ID NO: 1 is a substitution with serine, optionally V60S. Optionally, the mutation at a position corresponding to position 60 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally V60E.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine, glutamic acid, or phenylalanine. Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R383I. Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with leucine, optionally R383L.

Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R383Y. Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally R383E. Optionally, the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally R383F.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 384 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with isoleucine, tryptophan, tyrosine, or phenylalanine. Optionally, the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with isoleucine, optionally A3841. Optionally, the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with tryptophan, optionally A384W. Optionally, the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with tyrosine, optionally A384Y. Optionally, the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally A384F.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 400 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 400 of SEQ ID NO: 1 is a substitution with tryptophan, tyrosine, or arginine. Optionally, the mutation at a position corresponding to position 400 of SEQ ID NO: 1 is a substitution with tryptophan, optionally I400W. Optionally, the mutation at a position corresponding to position 400 of SEQ ID NO: 1 is a substitution with tyrosine, optionally I400Y. Optionally, the mutation at a position corresponding to position 400 of SEQ ID NO: 1 is a substitution with arginine, optionally MOOR.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 401 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with glycine, tyrosine, phenylalanine, or tryptophan. Optionally, the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with glycine, optionally H401G. Optionally, the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally H401Y. Optionally, the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally H401F. Optionally, the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tryptophan, optionally H401 W.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 407 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 407 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I407F.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 408 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 408 of SEQ ID NO: 1 is a substitution with threonine or tyrosine. Optionally, the mutation at a position corresponding to position 408 of SEQ ID NO: 1 is a substitution with threonine, optionally V408T. Optionally, the mutation at a position corresponding to position 408 of SEQ ID NO: 1 is a substitution with tyrosine, optionally V408Y. Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 409 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, tryptophan, or glutamic acid. Optionally, the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I409F. Optionally, the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with tryptophan, optionally I409W. Optionally, the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally I409E.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 439 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with leucine, glutamic acid, or glutamine. Optionally, the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with leucine, optionally glutamic acid G439L. Optionally, the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally G439E. Optionally, the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with glutamine, optionally G439Q.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 440 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 440 of SEQ ID NO: 1 is a substitution with threonine, optionally N440T.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 441 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with glutamic acid, phenylalanine, or tyrosine. Optionally, the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally K441E. Optionally, the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally K441F. Optionally, the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with tyrosine, optionally K441Y.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 479 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 479 of SEQ ID NO: 1 is a substitution with aspartic acid, optionally E479D.

Optionally, the one or more mutations of the modified CFI polypeptide comprises a mutation at a position corresponding to position 482 of SEQ ID NO: 1. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with tryptophan, leucine, histidine, isoleucine, glutamine, arginine, or threonine. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with tryptophan, optionally F482W. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with leucine, optionally F482L. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with histidine, optionally F482H. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with isoleucine, optionally F482I. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with glutamine, optionally F482Q. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with arginine, optionally F482R. Optionally, the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with threonine, optionally F482T.

Optionally, the one or more mutations of the modified CFI polypeptide comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 383 of SEQ ID NO: 1. Optionally:

(i) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R53I; and

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally R383E.

Optionally:

(i) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L; and

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, isoleucine, or tyrosine.

Optionally:

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R383I.

Optionally:

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R383Y.

Optionally:

(i) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y; and

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R383Y. Optionally, the one or more mutations of the modified CFI polypeptide comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 401 of SEQ ID NO: 1.

Optionally:

(ii) the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally H401Y.

Optionally:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 409 of SEQ ID NO: 1.

Optionally:

(ii) the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I409F.

Optionally:

(ii) the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I409F. Optionally, the one or more mutations of the modified CFI polypeptide comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 479 of SEQ ID NO: 1.

Optionally:

(ii) the mutation at a position corresponding to position 479 of SEQ ID NO: 1 is a substitution with aspartic acid, optionally E479D.

(i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 401 of SEQ ID NO: 1.

Optionally:

(i) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally R383F; and

(i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 409 of SEQ ID NO: 1.

Optionally:

Optionally, the one or more mutations of the modified CFI polypeptide comprises: (i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 439 of SEQ ID NO: 1. Optionally:

(ii) the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally G439E.

Optionally, the one or more mutations does not comprise a mutation selected from Ml 38V or S525A which are positions corresponding to positions 138 and 525, respectively, in SEQ ID NO: 1. Optionally, the one or more mutations does not comprise a M138V mutation (which is a position corresponding to position 138 in SEQ ID NO: 1).

Optionally, the one or more mutations does not comprise a S525A mutation (which is a position corresponding to position 525 in SEQ ID NO: 1). Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 380 of SEQ ID NO: 1, optionally at H380. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 429 of SEQ ID NO: 1, optionally at D429. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 525 of SEQ ID NO: 1, optionally at S525.

Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 53 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with A. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 383 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with A, V, M, W, G, P, S, T, N, Q, H, K, or D. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 383 of SEQ ID NO: 1, wherein the mutation is a substitution with A, V, I, M, W, G, P, S, T, N, Q, H, K, or D. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 383 of SEQ ID NO: 1, wherein the mutation is a substitution with A, V, I, M, W, G, P, S, T, N, Q, H, K, D, F, Y, L, or E. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 401 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 409 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 441 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G, A, or D. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 441 of SEQ ID NO: 1, wherein the mutation is a substitution with G, A, D, or E. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 384 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 400 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 407 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 408 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 409 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 440 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with K. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 441 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with G, A, E, or D. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 441 of SEQ ID NO: 1, wherein the mutation is a substitution with G, A, or D. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 479 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with Q, K, R, H, G, A, D, F, I, L, M, N, P, S, T, W, Y, or V. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 479 of SEQ ID NO: 1, wherein the mutation is a substitution with Q, K, R, H, G, A, F, I, L, M, N, P, S, T, W, Y, or V. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 482 of SEQ ID NO: 1, optionally wherein the mutation is a substitution with Y.

Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 482 of SEQ ID NO: 1, wherein the mutation is a substitution with Y, L, I, A, P, H, or G. Optionally, the one or more mutations does not comprise a mutation at a position corresponding to position 482 of SEQ ID NO: 1, wherein the mutation is a substitution with Y, A, P, or G.

Optionally, the one or more mutations does not comprise one or more of the modifications presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480 (incorporated herein by reference). Optionally, the one or more mutations does not comprise any one of the modifications presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480. Optionally, the one or more mutations does not comprise one or more of the modifications described in WO 2021/257480 (incorporated herein by reference). Optionally, the one or more mutations does not comprise any one of the modifications described in WO 2021/257480. Optionally, the one or more mutations does not comprise one or more of the substitution mutations presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480. Optionally, the one or more mutations does not comprise any one of the substitution mutations presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480. Optionally, the one or more mutations does not comprise one or more of the substitution mutations described in WO 2021/257480. Optionally, the one or more mutations does not comprise any one of the substitution mutations described in WO 2021/257480. Optionally, the modified CFI polypeptide does not comprise one or more of the CFI variants presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480. Optionally, the modified CFI polypeptide does not comprise any one of the CFI variants presented in Tables 2-9, 13, 15 and the Examples of WO 2021/257480. Optionally, the modified CFI polypeptide does not comprise one or more of the CFI variants described in WO 2021/257480. Optionally, the modified CFI polypeptide does not comprise any one of the CFI variants described in WO 2021/257480.

Optionally, the modified CFI polypeptide does not comprise an amino acid sequence encoded by one or more of the fusion constructs described in WO 2021/257480. Optionally, the modified CFI polypeptide does not comprise an amino acid sequence encoded by any one of the fusion constructs described in WO 2021/257480.

Optionally, the modified CFI polypeptide is not a fusion protein. In such embodiments, the term “fusion protein ” refers to a combination of a polypeptide component attached (e.g. directly or through a linker) to a CFI amino acid sequence. The “polypeptide component” is at least 80% identical to a contiguous fragment of a polypeptide which is not a CFI polypeptide, In such embodiments, the “contiguous fragment” is at least 25% of the “polypeptide which is not a CFI polypeptide” . For example, the “polypeptide which is not a CFI polypeptide ” may be albumin (such as human serum albumin) and, in such embodiments, the “polypeptide component” is at least 80% identical to a contiguous fragment of at least 25% of albumin (such as human serum albumin). In a further example, the “polypeptide component” may be a domain from the “polypeptide which is not a CFI polypeptide In a further example, the “polypeptide component” may be the “polypeptide which is not a CFI polypeptide” .

If the modified CFI polypeptide comprises a signal peptide which is not a wild type (native) CFI signal peptide, this does not render the modified CFI polypeptide a fusion protein. In particular, if the modified CFI polypeptide comprises a signal peptide which is from another polypeptide or is a synthetic signal peptide, this does not render the modified CFI polypeptide a fusion protein.

Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 1 or SEQ ID NO: 2 of at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, between 400 and 565 amino acids, between 500 and 565 amino acids, between 520 and 565 amino acids, between 400 and 583 amino acids, between 500 and 583 amino acids, or between 520 and 583 amino acids. Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2. Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to a fragment of between 500 and 583 amino acids of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to a fragment of between 500 and 565 amino acids of SEQ ID NO: 2. Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 2.

Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises the one or more mutations described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 60 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 384 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 400 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 401 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 407 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 408 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 409 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 439 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 440 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 441 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 479 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 482 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 383 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 401 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 and a mutation at a position corresponding to position 408 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 409 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 and a mutation at a position corresponding to position 439 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 479 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 and a mutation at a position corresponding to position 482 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 401 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 and a mutation at a position corresponding to position 408 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 409 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 439 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 and a mutation at a position corresponding to position 479 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 and a mutation at a position corresponding to position 482 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 408 and a mutation at a position corresponding to position 401 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 408 and a mutation at a position corresponding to position 479 of SEQ ID NO: 1 described herein. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 409 and a mutation at a position corresponding to position 479 of SEQ ID NO: 1 described herein.

Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with isoleucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with isoleucine at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with aspartic acid at a position corresponding to position 479 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 439 of SEQ ID NO: 1. Optionally, the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1. Optionally, the one or more mutations of the modified CFI polypeptide comprises the following combination of mutations selected from the positions described herein, wherein the positions correspond to their respective positions in SEQ ID NO: 1: R53I; R53L; R53Y; R53E; V60S; V60E; R383F; R383Y; R383I; R383L; R383E; A384I; A384W; A384Y; A384F; I400W; I400Y; MOOR; H401W; H401Y; H401F; H401G; I407F; V408T; V408Y; I409F; I409W; I409E; G439L; G439E; N440T; K441E; K441F; K441Y; E479D; F482W; F482L; F482H; F482I; F482Q; F482R; F482T; R53E and R383E; R53E and R383I; R53E and R383L; R53E and R383Y; R53E and R383F; R53E and V408T; R53E and I409F; R53E and G439Q; R53E and G439E; R53E and H401Y; R53E and E479D; R53I and R383E; R53I and R383I; R53I and R383L; R53I and R383Y; R53I and R383F; R53I and V408T; R53I and I409F; R53I and G439Q; R53I and G439E; R53I and H401Y; R53I and E479D; R53I and F482Q; R53L and R383E; R53L and R383I; R53L and R383L; R53L and R383Y; R53L and R383F; R53L and V408T; R53L and I409F; R53L and G439Q; R53L and G439E; R53L and H401Y; R53L and E479D; R53L and F482Q; R53Y and R383E; R53Y and R383I; R53Y and R383L; R53Y and R383Y; R53Y and R383F; R53Y and V408T; R53Y and I409F; R53Y and G439Q; R53Y and G439E; R53Y and H401Y; R53Y and E479D; R53Y and F482Q; R383E and V408T; R383E and I409F; R383E and G439E; R383E and H401Y; R383E and E479D; R383I and V408T; R383I and I409F; R383I and G439Q; R383I and G439E; R383I and H401Y; R383L and I409F;

R383L and G439E; R383L and H401Y; R383L and E479D; R383L and F482Q; R383Y and V408T; R383Y and I409F; R383Y and G439Q; R383Y and G439E; R383Y and H401Y; R383Y and E479D; R383Y and F482Q; R383F and V408T; R383F and I409F; R383F and G439Q; R383F and G439E; R383F and H401Y; R383F and E479D; R383F and F482Q; V408T and H401Y; V408T and E479D; I409F and E479D; G439Q and H401Y; G439Q and E479D; G439Q and F482Q; G439E and H401Y; G439E and E479D; G439E and F482Q; R53E, R383E,and I409F; R53E, R383E, and G439E; R53E, R383E, and H401Y; R53L, R383L, and V408T; R53L, R383L, and G439E; R53L, R383L, and E479D; R383E, G439Q, and V408T; R383E, G439Q, and I409F; R383E, G439Q, and H401Y; R383E, G439Q, and F482Q; V408T, I409F, and G439Q; V408T, I409F, and H401Y; V408T, I409F, and E479D; V408T, I409F, and F482Q; H401Y, E479D, and F482Q; R53E, R383E, I409F, and G439E; R53E, R383E, V408T, and F482Q; R53E, R383E, I409F, and H401Y; R53E, R383E, G439E, and H401Y; R53E, R383L, V408T, and H401Y; R53E, R383L, V408T, and F482Q; R53E, R383L, G439E, and F482Q; R53E, R383Y, V408T, and H401Y; R53E, R383Y, V408T, and F482Q; R53E, R383Y, I409F, and H401Y; R53E, R383Y, I409F, and F482Q; R53E, R383F, V408T, and F482Q; R53E, R383F, I409F, and H401Y; R53E, R383F, I409F, and F482Q; R53E, R383F, G439E, and H401Y; R53E, R383F, G439E, and F482Q; R53I, R383E, G439E, and H401Y; R53I, R383I, V408T and H401Y; R53I, R383I, V408T and F482Q; R53I, R383I, G439Q, and H401Y; R53I, R383I, G439Q, and F482Q; R53I, R383L, V408T, and H401Y; R53I, R383L, G439Q, and H401Y; R53I, R383L, G439Q, and F482Q; R53I, R383Y, V408T, and H401Y; R53I, R383Y, V408T, and F482Q; R53I, R383Y, G439Q, and H401Y; R53I, R383Y, G439Q, and F482Q; R53I, R383F, V408T, and H401Y; R53I, R383F, V408T, and F482Q; R53I, R383F, G439Q, and H401Y; R53I, R383F, G439Q, and F482Q; R53L, R383E, V408T, and H401Y; R53L, R383E, I409F, and F482Q; R53L, R383E, G439E, and H401Y; R53L, R383E, G439E, and F482Q; R53L, R383I, V408T, and H401Y;

R53L, R383I, G439Q, and H401Y; R53L, R383L, V408T, and H401Y; R53L, R383L, V408T , and F482Q; R53L, R383L, G439Q, and H401Y; R53L, R383L, G439E, and F482Q; R53L, R383Y, V408T, and H401Y; R53L, R383Y, V408T, and F482Q; R53L, R383Y, I409F, and H401 Y; R53L, R383Y, I409F, and F482Q; R53L, R383Y, G439Q, and H401Y; R53L, R383Y, G439Q, and F482Q; R53L, R383Y, G439E, and H401Y; R53L, R383F, V408T, and H401Y; R53L, R383F, V408T, and F482Q; R53L, R383F, I409F, and H401Y; R53L, R383F, I409F, and F482Q; R53L, R383F, G439Q, and H401Y; R53L, R383F, G439Q, and F482Q; R53L, R383F, G439E, and H401Y; R53L, R383F G439E, and F482Q; R53Y, R383E, I409F, and F482Q; R53Y, R383E, G439E, and H401Y; R53Y, R383E, G439E, and F482Q; R53Y, R383L, V408T, and H401Y; R53Y, R383L, V408T, and F482Q; R53Y, R383L, I409F, and H401Y; R53Y, R383L, G439Q, and H401Y; R53Y, R383L, G439E, and F482Q; R53Y, R383Y, V408T, and H401Y; R53Y, R383Y, V408T, and F482Q; R53Y, R383Y, I409F, and H401Y; R53Y, R383Y, I409F, and F482Q; R53Y, R383Y, G439Q, and H401Y; R53Y, R383Y, G439Q, and F482Q; R53Y, R383F, V408T, and H401Y; R53Y, R383F, V408T, and F482Q; R53Y, R383F, I409F, and H401 Y; R53Y, R383F, I409F, and F482Q; R53Y, R383F, G439Q, and H401Y; R53Y, R383F, G439Q and F482Q; R53Y, R383F, G439E, and H401Y; R53Y, R383F, G439E, and F482Q; R53Y, H401Y, I409F, E479D, and F482Q; R383E, H401Y, I409F, E479D, and F482Q; R383I, H401Y, I409F, E479D, and F482Q; R383L, H401Y, I409F, E479D, and F482Q; and R383Y, H401Y, I409F, E479D, and F482Q.

Optionally, the one or more mutations of the modified CFI polypeptide comprises the following combination of mutations selected from the positions described herein, wherein the positions correspond to their respective positions in SEQ ID NO: 1: R53Y, R383I, V408T and H401Y; R53Y, R383I, G439Q and H401 Y; and R53Y, R383I, G439Q and F482Q.

In one embodiment, the at least one mutation comprises: i) a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L; and

(ii) a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R383Y, and wherein the modified CFI polypeptide has at least 5 fold higher CFI activity compared to a reference CFI polypeptide when measured after 30 minutes incubation at pH 7 with C3b and CFH, followed by 30 minutes of incubation with an anti-iC3b (Tb) IgG and an anti-C3b (FITC) IgG, optionally wherein the reference CFI polypeptide is a wild -type CFI polypeptide.

A polynucleotide comprising a CFI nucleotide sequence

The invention provides a polynucleotide comprising a CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes the modified CFI polypeptide of the invention. The nucleotide sequence has at least one difference compared to a wild-type CFI nucleotide sequence, e.g. a mutation has been introduced.

The terms “nucleic acid molecule ”, “polynucleotide ” and “nucleotide sequence ” are intended to refer to a polymeric chain of any length of nucleotides, including deoxyribonucleotides, ribonucleotides, or analogs thereof. For example, the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The nucleic acid molecule, polynucleotide or nucleotide sequence may consist of DNA. The nucleic acid molecule, polynucleotide or nucleotide sequence may be mRNA. Since the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

In some embodiments, the term “nucleotide sequence ” can be replaced with the term “nucleic acid molecule” .

A CFI nucleotide sequence encodes a CFI polypeptide. The polynucleotide of the invention comprises a CFI nucleotide sequence and the CFI nucleotide sequence encodes the modified CFI polypeptide of the invention. The term “sequence that encodes ” or “sequence encodes ” refers to a nucleotide sequence comprising an open reading frame comprising codons that encode the encoded polypeptide. For example, a nucleotide sequence that encodes a CFI polypeptide comprises codons that encode the amino acid sequence of the CFI polypeptide. An example of a CFI nucleotide sequence that encodes a wild-type CFI is provided in SEQ ID NO: 3, 4, 28, 18 or 19.

The codons that encode the polypeptide are also referred to as “coding nucleotides ”. A CFI nucleotide sequence may be interrupted by non-coding nucleotides (e.g. an intron), but only nucleotides that encode the polypeptide (i.e. the coding nucleotides) should be considered to be part of the CFI nucleotide sequence. For example, a CFI nucleotide sequence that encodes a CFI polypeptide will comprise any codons (i.e. the coding nucleotides) that encode an amino acid forming part of the CFI polypeptide, irrespective of whether those codons are contiguous in sequence or separated by one or more non-coding nucleotides. In other words, a CFI polynucleotide which contains stretches of coding nucleotides interrupted by a stretch of non-coding nucleotides will be considered to comprise a “CFI nucleotide sequence” consisting of the non-contiguous coding stretches immediately juxtaposed (j.e. minus the stretches of non-coding nucleotides). However, herein, the nucleotides of the stop codon will be considered coding nucleotides.

A CFI nucleotide sequence encoding a CFI polypeptide may also comprise a sequence that encodes a signal peptide. It is well known that some proteins, particularly those which are exported to different tissues, are expressed with a signal peptide. Signal peptides can be at the N-terminus of a protein sequence (and in this case at the 5’ end of a coding sequence) and many signal peptides are cleaved following cellular processing. Thus, herein, a mature protein or polypeptide (such as a mature CFI protein or polypeptide) will be considered to be the resulting protein or polypeptide after the signal peptide has been processed and removed/cleaved (and thus no longer forms part of the polypeptide sequence). The modified CFI polypeptide may be “furin-cleaved”, i.e. may be cleaved at a furin cleavage site into a heavy and a light chain. In such cases, the modified CFI polypeptide comprises a disulphide-linked dimer of the CFI heavy chain (corresponding to residues 19-335 of SEQ ID NO: 1) and the light chain (corresponding to residues 340-583 of SEQ ID NO: 1).

The following Table describes codons that encode each amino acid:

Table A

The corresponding RNA codons will contain Us in place of the Ts in the Table above.

The present invention provides a polynucleotide comprising a modified CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes a modified CFI polypeptide of the invention. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%. at least 99.8%, at least 99.9% or 100% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 16, 17, 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 16, 17, 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%. at least 99.8%, at least 99.9% or 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 16, 17, 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a nucleotide sequence of any one of SEQ ID NOs: 16, 17, 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 16 or 17 are replaced with references to SEQ ID NOs: 28 or 29. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of SEQ ID NO: 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1500 nucleotides of SEQ ID NO: 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a nucleotide sequence of SEQ ID NO: 18 or 19. Optionally, the CFI nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 18 or 19.

Optionally, the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 18 or 19, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1 described herein. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R53Y. For example, the codon may encode a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with phenylalanine, optionally R383F. For example, the codon may encode a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R383Y. For example, the codon may encode a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with leucine, optionally R383L. For example, the codon may encode a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with glutamic acid, optionally R383E. For example, the codon may encode a mutation at a position which corresponds to position 401 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally H401Y. For example, the codon may encode a mutation at a position which corresponds to position 401 of SEQ ID NO: 1 and is a substitution with phenylalanine, optionally H401F. For example, the codon may encode a mutation at a position which corresponds to position 401 of SEQ ID NO: 1 and is a substitution with glycine, optionally H401G. For example, the codon may encode a mutation at a position which corresponds to position 409 of SEQ ID NO: 1 and is a substitution with tryptophan, optionally 1409 W. For example, the codon may encode a mutation at a position which corresponds to position 409 of SEQ ID NO: 1 and is a substitution with glutamic acid, optionally I409E. For example, the codon may encode a mutation at a position which corresponds to position 439 of SEQ ID NO: 1 and is a substitution with glutamic acid, optionally G439E. For example, the codon may encode a mutation at a position which corresponds to position 441 of SEQ ID NO: 1 and is a substitution with glutamic acid, optionally K441E. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with isoleucine, optionally R53I, and a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with glutamic acid, optionally R383E. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L, and a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with isoleucine, optionally R383I. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L, and a mutation at a position which corresponds to position 383 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R383Y. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L, and a mutation at a position which corresponds to position 401 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally H401Y. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R53Y, and a mutation at a position which corresponds to position 409 of SEQ ID NO: 1 and is a substitution with phenylalanine, optionally I409F. For example, the codon may encode a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally R53Y, and a mutation at a position which corresponds to position 401 of SEQ ID NO: 1 and is a substitution with tyrosine, optionally H401Y.

Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 30 or 31. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 32 or 33. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 34 or 35. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 36 or 37. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 38 or 39. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 40 or 41. Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 42 or 43.

Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NOs: 18 or 19 are replaced with references to SEQ ID NOs: 44, 45, 46, 47, 48, 49, 50, 51, or 52.

Optionally, the CFI nucleotide sequence comprises a sequence as described above, except that references to SEQ ID NO: 18 or 19 is replaced with references to SEQ ID NO: 4 or 3.

Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations described herein. Optionally, the CFI nucleotide sequence is a variant of the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515 encoding a CFI polypeptide comprising a sequence at least 95% identical to the polypeptide encoded by SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from W02017/07251, respectively. Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. Unless a sequence identifier is otherwise specified as being from a particular source, e.g. SEQ ID NO: 1 from W02020/086735, reference to a particular sequence identifier herein should be understood to represent a sequence as described herein and listed in the “Description of the Sequence Listing” and Figure 6. For SEQ ID NOs: 28 to 56, reference to a particular sequence identifier herein should be understood to represent a sequence as described herein and listed in the “Description of the Sequence Listing”. Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide has one or more, two or more, three of more, four or more, or five or more amino acid substitutions at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. For example, the encoded CFI polypeptide has (i) a substitution with isoleucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53I; and (ii) a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383E. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with isoleucine at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383I. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383Y. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1, optionally H401Y. For example, the encoded CFI polypeptide has (i) a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53Y; and (ii) a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1, optionally I409F. For example, the encoded CFI polypeptide has (i) a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53Y; and (ii) a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1, optionally H401Y.

Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 44 to 52, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations described herein. Optionally, the CFI nucleotide sequence is a variant of the nucleotide sequence of any one of SEQ ID NOs: 44 to 52 encoding a CFI polypeptide comprising a sequence at least 95% identical to the polypeptide encoded by SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, or 52, respectively. Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 44 to 52, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. Optionally, the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 44 to 52, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide has one or more, two or more, three of more, four or more, or five or more amino acid substitutions at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1. For example, the encoded CFI polypeptide has (i) a substitution with isoleucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53I; and (ii) a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383E. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with isoleucine at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383I. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383Y. For example, the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1, optionally H401Y. For example, the encoded CFI polypeptide has (i) a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53Y; and (ii) a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1, optionally I409F. For example, the encoded CFI polypeptide has (i) a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53Y; and (ii) a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1, optionally H401Y.

In an embodiment, at least a portion of the CFI nucleotide sequence is codon-optimised. Optionally, all or a portion of the CFI nucleotide sequence is codon-optimised. Codonoptimisation can improve expression of the nucleotide sequence, for example a CFI nucleotide sequence, in a particular tissue and/or in a particular organism. For example, if a nucleotide sequence is codon-optimised for expression in the human liver, the nucleotide sequence may be modified to increase the number of codons that may be favoured (in the sense that such codons correspond to tRNA species which are more abundant than other tRNA species specific for the same amino acid) in the human liver. As a further example, if a nucleotide sequence is codon-optimised for expression in the human liver cells, the nucleotide sequence may be modified to increase the number of codons that may be favoured (in the sense that such codons correspond to tRNA species which are more abundant than other tRNA species specific for the same amino acid) in human liver cells. Codon-optimisation can improve expression of the nucleotide sequence, for example a CFI nucleotide sequence, in a particular cell line. For example, if a nucleotide sequence is codon-optimised for expression in Chinese hamster ovary (CHO) (or derivative) cells, the nucleotide sequence may be modified to increase the number of codons that may be favoured (in the sense that such codons correspond to tRNA species which are more abundant than other tRNA species specific for the same amino acid) in such CHO cells. The skilled person would appreciate that codon-optimising a sequence may not entail changing every codon, not least because a ‘favoured codon’'’ may already be present at some positions.

Such codon-optimisation may be subject to other factors. For example, favoured codons may not be introduced at positions where doing so introduces CpGs into the sequence; this will still be considered to be codon-optimisation. In an embodiment, a favoured codon that ends with a C nucleotide will not be included in the portion of the coding sequence that is codon-optimised, where the next codon in the sequence begins with a G. For example, codon GCC encodes alanine. Optionally, where GCC is a favoured codon, it should not be used for encoding alanine where the next codon in the sequence begins with a G, such as codon GAC (or alternatively, the next codon - where possible - could be selected to avoid a G at the first position).

It is straightforward to determine the frequency of each codon used in a portion of a nucleotide sequence. The skilled person merely needs to enter the sequence of that portion into one of the readily-available algorithms that looks at codon usage and review the results. Alternatively, the user could simply count them.

Optionally, the portion of the CFI nucleotide sequence that is codon-optimised is codon- optimised for expression in human liver cells. Optionally, the portion of the CFI nucleotide sequence that is codon-optimised is a contiguous portion. Optionally, the CFI nucleotide sequence is codon-optimised for expression in human liver cells. Optionally, the CFI nucleotide sequence or the portion thereof is codon optimised for expression in human liver cells if the CFI nucleotide sequence expresses the CFI polypeptide or the fragment thereof in human liver cells at a higher level compared to expression of a CFI polypeptide encoded by a reference CFI nucleotide sequence. Optionally, the CFI nucleotide sequence or the portion thereof is codon optimised for expression in human liver cells if the CFI nucleotide sequence expresses the CFI polypeptide or the fragment thereof in human liver cells at a higher level on transfection of the polynucleotide comprising the CFI nucleotide sequence compared to expression of the CFI polypeptide encoded by the reference CFI nucleotide sequence on transfection with an equivalent polynucleotide comprising the reference CFI nucleotide sequence. In such embodiments, the reference CFI nucleotide sequence may be a wild-type CFI nucleotide sequence. The wild type CFI nucleotide may be the sequence of SEQ ID NO: 3 or 4. The "''equivalent” polynucleotide comprising the reference CFI nucleotide is a polynucleotide which is identical (i.e. comprises the same transcription regulatory elements etc.) to the polynucleotide of the invention except that the CFI nucleotide sequences are different. For example, the different CFI nucleotide sequences being compared are operably linked to the same promoter sequence. Optionally, the human liver cell is a Huh-7 cell.

Optionally, the portion of the CFI nucleotide sequence that is codon-optimised is codon- optimised for expression in human retinal cells or human kidney cells. Thus, optionally, the references to human liver cells above may be replaced with references to human retinal cells or human kidney cells. For example, optionally, the CFI nucleotide sequence is codon-optimised for expression in human retinal cells or human kidney cells.

The presence of CpGs (z.e. CG dinucleotides) in a gene therapy vector may have an adverse effect on expression of a therapeutic transgene (e.g. the durability of said expression). This is because CpGs may be methylated, and their methylation may lead to gene silencing thereby reducing expression. Also, it is possible that high CpG content could trigger a TLR response, increasing the risk of an anti- AAV immune response. The CFI nucleotide sequence or portion of the CFI nucleotide sequence that is codon-optimised may comprise a reduced number of CpGs compared to a corresponding portion of a reference CFI nucleotide sequence. The reference CFI nucleotide sequence may be a wildtype CFI nucleotide sequence. The wild type CFI nucleotide may be the sequence of SEQ ID NO: 3 or 4. The reference CFI nucleotide sequence may be the nucleotide sequence of SEQ ID NO: 3. The phrase “a corresponding portion of a reference CFI nucleotide sequence ” is intended to refer to the portion of the reference CFI nucleotide sequence which “corresponds to ” the CFI nucleotide sequence or portion of the CFI nucleotide sequence that is codon-optimised. It is within the capabilities of the person skilled in the art to determine the portion of the reference CFI nucleotide sequence which “corresponds to ” the CFI nucleotide sequence or portion of the CFI nucleotide sequence that is codon- optimised. For example, the person skilled in the art merely needs to perform a sequence alignment of the reference CFI nucleotide sequence with the CFI nucleotide sequence of the invention using a suitable alignment algorithm such as that of Needleman and Wunsch described above, and determine which region of the reference CFI nucleotide sequence aligns with the CFI nucleotide sequence or portion of the CFI nucleotide sequence that is codon-optimised.

In an embodiment, the CFI nucleotide sequence or portion of the CFI nucleotide sequence that is codon-optimised (which may be all of the CFI nucleotide sequence) comprises 40 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer CpGs. In an embodiment, the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon- optimised (which may be all of the CFI nucleotide sequence) comprises 5 or fewer CpGs. In an embodiment, the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised (which may be all of the CFI nucleotide sequence) comprises 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer CpGs per 100 nucleotides. In some embodiments, the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised (which may be all of the CFI nucleotide sequence) is CpG-free, i.e. contains no (0) CG dinucleotides.

The CFI nucleotide sequence may encode a signal peptide. Optionally, the amino acid sequence of the signal peptide is an amino acid sequence of a wild type CFI signal peptide. The amino acid sequence of a wild type CFI signal peptide may be the amino acid sequence of SEQ ID NO: 6. The signal peptide may be encoded by a nucleotide sequence of SEQ ID NO: 5. The signal peptide may not be encoded by a wild-type CFI signal peptide-encoding nucleotide sequence. The signal peptide may be encoded by nucleotides 1 to 54 of any one of SEQ ID NOs: 3, 16, 17, and 19. Optionally, the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide. For example, the signal peptide may not be a wild type CFI signal peptide. The signal peptide may be the signal peptide from another polypeptide. The signal peptide may be a heterologous signal peptide. The heterologous signal peptide may be, for example, a signal peptide from another polypeptide, a modified CFI signal peptide, or a synthetic signal peptide. A “modified CFI signal peptide” is a wild type CFI signal peptide which has been modified (e.g. a mutation has been introduced). Optionally, the CFI nucleotide sequence encodes a signal peptide. Optionally, the CFI nucleotide sequence does not encode a signal peptide. Optionally, the CFI polypeptide or fragment thereof comprises a signal peptide. Optionally, the CFI polypeptide or fragment thereof does not comprise a signal peptide.

The polynucleotide of the invention may comprise a transcription regulatory element.

Any appropriate transcription regulatory element may be used, such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, or LSP, which are all liver-specific transcription regulatory elements. These transcription regulatory elements are described in more detail in the following references: HLP1: McIntosh J. et al., Blood 2013 Apr 25, 121 (17):3335-44; LP1: Nathwani et al., Blood. 2006 April 1, 107(7): 2653-2661; HCR-hAAT: Miao et al., Mol Ther. 2000; 1: 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637- 645 (1996); and LSP: Wang et al., Proc Natl Acad Sci U S A. 1999 March 30, 96(7): 3906-3910. The transcription regulatory element may comprise a liver-specific promoter.

The transcription regulatory element may comprise a promoter and/or an enhancer, such as the promoter element and/or enhancer element from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, or LSP. Each of these transcription regulatory elements comprises a promoter, an enhancer, and optionally other nucleotides. The transcription regulatory element may be operably linked to the CFI nucleotide sequence of the invention. The promoter and/or enhancer may operably linked to the CFI nucleotide sequence of the invention.

Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 500 bp, at least 1000 bp, at least 1400 bp, or at least 1450 bp of SEQ ID NO: 11. Optionally, the transcription regulatory element is at least 98%, at least 99%, or 100% identical to a fragment of at least 1400 bp or at least 1450 bp of SEQ ID NO: 11. Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the transcription regulatory element is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98% identical to SEQ ID NO: 11. Optionally, the polynucleotide comprises a transcription regulatory element that has a sequence of SEQ ID NO: 11. Optionally, the polynucleotide comprises a transcription regulatory element of SEQ ID NO: 11. Optionally, the polynucleotide comprises a transcription regulatory element consisting of SEQ ID NO: 11.

Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 320 bp, or at least 330 bp of SEQ ID NO: 20. Optionally, the transcription regulatory element is at least 98%, at least 99%, or at least 100% identical to a fragment of at least 300 bp or at least 320 bp of SEQ ID NO: 20. Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 20. Optionally, the transcription regulatory element is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 20. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98% identical to SEQ ID NO: 20. Optionally, the polynucleotide comprises a transcription regulatory element that has a sequence of SEQ ID NO: 20. Optionally, the polynucleotide comprises a transcription regulatory element of SEQ ID NO: 20. Optionally, the polynucleotide comprises a transcription regulatory element consisting of SEQ ID NO: 20.

Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 720 bp, or at least 730 bp of SEQ ID NO: 21. Optionally, the transcription regulatory element is at least 98%, at least 99%, or at least 100% identical to a fragment of at least 700 bp or at least 720 bp of SEQ ID NO: 21. Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 21. Optionally, the transcription regulatory element is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 21. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98% identical to SEQ ID NO: 21. Optionally, the polynucleotide comprises a transcription regulatory element that has a sequence of SEQ ID NO: 21. Optionally, the polynucleotide comprises a transcription regulatory element of SEQ ID NO: 21. Optionally, the polynucleotide comprises a transcription regulatory element consisting of SEQ ID NO: 21.

Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, or at least 100 bp of SEQ ID NO: 22. Optionally, the transcription regulatory element is at least 98%, at least 99%, or at least 100% identical to a fragment of at least 90 bp or at least 100 bp of SEQ ID NO: 22. Optionally, the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 22. Optionally, the transcription regulatory element is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 22. Optionally, the polynucleotide comprises a transcription regulatory element that is at least 98% identical to SEQ ID NO: 22. Optionally, the polynucleotide comprises a transcription regulatory element that has a sequence of SEQ ID NO: 22. Optionally, the polynucleotide comprises a transcription regulatory element of SEQ ID NO: 22. Optionally, the polynucleotide comprises a transcription regulatory element consisting of SEQ ID NO: 22.

The transcription regulatory element may comprise an ocular-specific promoter and/or enhancer. Optionally, the transcription regulatory element is an ocular-specific transcription regulatory element. Optionally, the transcription regulatory element comprises a retinal cell-specific promoter and/or enhancer. Optionally, the transcription regulatory element is a retinal cell-specific transcription regulatory element. Optionally, the transcription regulatory element comprises or is a rhodopsin promoter, a rhodopsin kinase promoter (Allocca et al. (2007) J. Virol. 81: 1 1372-80), pR2.1 (Mancuso et al. (2009) Nature 461: 784-7), a RPE65 promoter (Bainbridge et al. (2008) N. Engl. J. Med. 358: 2231 -9), or a VMD2 promoter (Esumi et al. (2004) J. Biol. Chem. 279: 19064-73).

The transcription regulatory element may comprise a kidney-specific promoter and/or enhancer. Optionally, the transcription regulatory element is a kidney-specific transcription regulatory element. Optionally, the transcription regulatory element comprises a podocyte-specific promoter and/or enhancer. Optionally, the transcription regulatory element is a podocyte-specific transcription regulatory element. Optionally, the transcription regulatory element comprises or is a podocyte-specific promoter. For example, the transcription regulatory element comprises or is a minimal NPHS1 promoter, a minimal NPHS2 promoter, or an alternative minimal NPHS 1 promoter. Optionally, the transcription regulatory element is at least 80%, at least 90%, at least 95%, at least 99.5%, at least 99.8%, or 100% identical to any one of SEQ ID NOs: 54, 55, or 56.

The transcription regulatory element may be a non-tissue-specific transcription regulatory element. In other words, the transcription regulatory element may be an ubiquitous transcription regulatory element. Optionally, the transcription regulatory element comprises a cytomegalovirus (CMV) promoter. Optionally, the transcription regulatory element comprises a chicken beta-actin (CBA) promoter. Optionally, the transcription regulatory element comprises the promoter element and/or enhancer element from a CAG transcription regulatory element (chicken beta-actin (CBA) promoter in combination with a cytomegalovirus (CMV) enhancer element). Optionally, the transcription regulatory element is a CAG transcription regulatory element. Optionally, the transcription regulatory element is at least 80%, at least 90%, at least 95%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 53.

In an embodiment, the polynucleotide of the invention comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) or a variant thereof. For example, the polynucleotide of the invention comprises the mutated WPRE sequence described in Zanta-Boussif et al (2009), Gene Therapy, 16:605-619. Optionally, the woodchuck hepatitis post-transcriptional regulatory element is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to any one of SEQ ID NOs: 23 to 25. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to any one of SEQ ID NOs: 23 to 25. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element that is at least 98% identical to any one of SEQ ID NOs: 23 to 25. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element that has a sequence of any one of SEQ ID NOs: 23 to 25. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element of any one of SEQ ID NOs: 23 to 25. Optionally, the polynucleotide comprises a woodchuck hepatitis post-transcriptional regulatory element consisting of any one of SEQ ID NOs: 23 to 25. In another embodiment, the polynucleotide of the invention does not comprise a woodchuck hepatitis posttranscriptional regulatory element (WPRE).

In some embodiments, the polynucleotide of the invention comprises a posttranscriptional regulatory element (PRE), wherein the PRE is not a WPRE. The polynucleotide of the invention may further comprise one or two ITRs. In one embodiment, the nucleotide sequence of the or each ITR is fewer than 157, fewer than 154, or around 145 nucleotides in length. Optionally, the or each ITR is a wild-type ITR. Optionally, the or each ITR is an AAV2 ITR. In some embodiments, the nucleotide sequence of the or each ITR comprises a nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

The polynucleotide of the invention may further comprise a polyA nucleotide sequence. The poly A sequence may be a bovine growth hormone poly A sequence (bGHpA - SEQ ID NO: 14). In some embodiments, the polyA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 14. The poly A sequence may be between 200 to 220, between 205 to 215, or around 208 nucleotides in length. The poly A sequence may be positioned downstream of the CFI nucleotide sequence of the invention.

The polynucleotide of the invention may further comprise an intron sequence, such as a viral intron sequence, optionally an SV40 intron sequence (SEQ ID NO: 15).

A viral particle comprising the polynucleotide

The invention further provides a viral particle comprising a recombinant genome comprising the polynucleotide of the invention. For the purposes of the present invention, the term “viral particle” refers to all or part of a virion. For example, the viral particle comprises a recombinant genome and may further comprise a capsid. The viral particle may be a gene therapy vector. Herein, the terms “viral particle” and “vector” are used interchangeably. For the purpose of the present application, a “gene therapy” vector is a viral particle that can be used in gene therapy, i.e. a viral particle that comprises all the required functional elements to express a transgene, such as a CFI nucleotide sequence, in a host cell after administration. Suitable viral particles include a parvovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adeno-associated virus (AAV). Optionally, the viral particle is an AAV, adenoviral or lentiviral viral particle. The viral particle is preferably a recombinant AAV vector or a lentiviral vector. More preferably, the viral particle is an AAV viral particle. The terms AAV and rAAV are used interchangeably herein, unless context obviously suggests otherwise.

The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is fewer than about 5,000 nucleotides in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal -145 nt (ITRs) are self- complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild type (wt) AAV infection in mammalian cells the Rep genes (i.e. encoding Rep78 and Rep52 proteins) are expressed from the P5 promoter and the Pl 9 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.

As described above, the polynucleotide of the invention may comprise one or two ITR(s). Thus, the recombinant viral genome may comprise one or two ITR(s) as described above. It is possible for an AAV vector of the invention to function with only one ITR. Thus, the viral genome typically comprises at least one ITR, but, more typically, two ITRs (generally with one either end of the viral genome, i.e. one at the 5’ end and one at the 3’ end). There may be intervening sequences between the CFI nucleotide sequence of the invention and one or both of the ITRs. The CFI nucleotide sequence may be incorporated into a viral particle located between two regular ITRs or located on either side of an ITR engineered with two D regions.

AAV sequences that may be used in the present invention for the production of AAV vectors can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number U89790; GenBank Accession number JO 1901 ; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al, 1999; Rutledge et al, 1998; and Wu et al, 2000. AAV serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11 or 12 may be used in the present invention. The sequences from the AAV serotypes may be mutated or engineered when being used in the production of gene therapy vectors.

Optionally, an AAV vector comprises ITR sequences which are derived from AAV 1 , AAV2, AAV4 and/or AAV6. Preferably the ITR sequences are AAV2 ITR sequences. Herein, the term AAVx/y refers to a viral particle that comprises genomic components such as at least ITRs from AAVx (wherein x is a AAV serotype number) and has the capsid from AAVy (wherein y is the number of the same or different serotype). For example, an AAV2/8 vector may comprise a portion of a viral genome, including the ITRs, from an AAV2 strain, and a capsid from an AAV8 strain.

In an embodiment, the viral particle comprises a capsid. In an embodiment, the viral particle is an AAV viral particle comprising a capsid. AAV capsids are generally formed from three proteins, VP1, VP2 and VP3. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 which does not form part of VP2 is referred to as VPlunique or VP1U. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 which does not form part of VP3 is referred to as VP2unique or VP2U. Optionally, the viral particle comprises a liver-tropic capsid. Whether a viral particle (capsid) is tropic for a particular tissue can be evaluated for example by administering such a particle expressing a marker gene such as luciferase and imaging in vivo at multiple time points (for example as described in Zincarelli et al (2008), Molecular Therapy, 16:1073- 1080). A particle driving strong marker expression in liver tissues, especially if in contrast to lesser expression in other tissues, would be considered liver-tropic. Optionally, the viral particle comprises a kidney-tropic capsid.

In some embodiments, a liver-tropic capsid can be an AAV3-, AAV3B-, AAV5-, or AAV8-derived capsid. Optionally, the liver-tropic capsid can be an AAV3-, AAV3B-, or AAV8-derived capsid. Optionally, the liver-tropic capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and 734, between 700 and 736, around 734, or around

736 amino acids of any one of SEQ ID NOs: 7-10. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to any one of SEQ ID NOs: 7-10. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to SEQ ID NO: 7. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to SEQ ID NO: 8. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to

SEQ ID NO: 9. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to SEQ ID NO: 10. Optionally, the liver-tropic capsid comprises a sequence 100% identical to SEQ ID NO: 7. Optionally, the liver-tropic capsid comprises a sequence 100% identical to SEQ ID NO: 8. Optionally, the liver-tropic capsid comprises a sequence 100% identical to SEQ ID NO: 9. Optionally, the liver-tropic capsid comprises a sequence

100% identical to SEQ ID NO: 10.

In some embodiments, a capsid can be an AAV3-, AAV3B-, AAV5-, or AAV8-derived capsid. Optionally, the capsid can be an AAV3-, AAV3B-, or AAV8-derived capsid. Optionally, the capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and

734, between 700 and 736, around 734, or around 736 amino acids of any one of SEQ ID NOs: 7-10. Optionally, the capsid comprises a sequence at least 99% identical to any one of SEQ ID NOs: 7-10. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 7. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 8. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 9. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 10. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 7. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 8. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 9. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 10.

In some embodiments, the capsid can be an AAV9-derived capsid. Optionally, the capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and 734, between 700 and 736, around 734, or around 736 amino acids of SEQ ID NO: 26. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 26. Optionally, the livertropic capsid comprises a sequence 100% identical to SEQ ID NO: 26. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 26.

In some embodiments, the capsid can be an AAV2 -derived capsid. Optionally, the capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and

735, between 650 and 734, between 650 and 735, between 700 and 734, between 700 and 735, around 734, or around 735 amino acids of SEQ ID NO: 27. Optionally, the capsid comprises a sequence at least 99% identical to SEQ ID NO: 27. Optionally, the livertropic capsid comprises a sequence 100% identical to SEQ ID NO: 27. Optionally, the capsid comprises a sequence 100% identical to SEQ ID NO: 27. In some embodiments, the capsid is not an AAV2-derived capsid. In some embodiments, the capsid is not an AAV8-derived capsid.

A viral particle of the invention may be a "hybrid" particle in which the viral ITRs and viral capsid are from different parvoviruses, such as different AAV serotypes. Preferably, the viral ITRs and capsid are from different serotypes of AAV, in which case such viral particles are known as transcapsidated or pseudotyped. Likewise, the parvovirus may have a "chimeric" capsid (e. g., containing sequences from different parvoviruses, preferably different AAV serotypes) or a "targeted" capsid (e. g., a directed tropism).

In some embodiments, the recombinant AAV genome comprises intact ITRs, comprising functional terminal resolution sites (TRS). Such an AAV genome may contain one or two resolvable ITRs, i.e. ITRs containing a functional TRS at which site-specific nicking can take place to create a free 3 ’ hydroxyl group which can serve as a substrate for DNA polymerase to unwind and copy the ITR.

Preferably, the recombinant genome is single-stranded (i.e., it is packaged into the viral particle in a single-stranded form). Optionally, the recombinant genome is not packaged in self-complementary configuration, i.e. the genome does not comprise a single covalently- linked polynucleotide strand with substantial self-complementary portions that anneal in the viral particle. Alternatively, the recombinant genome may be packaged in '"monomeric duplex” form. “Monomeric duplexes” are described in WO 2011/122950. The genome may be packaged as two substantially complementary but non-covalently linked polynucleotides which anneal in the viral particle.

As described above, the polynucleotide of the invention may comprise polyA nucleotide sequence. Thus, the viral particle may comprise the poly A sequence as described above. As described above, the polynucleotide of the invention may comprise an intron sequence. Thus, the viral particle of the invention may comprise the intron sequence as described above.

As described above, the polynucleotide of the invention may comprise one or two ITRs. Thus, the viral particle of the invention may comprise one or two ITRs as described above.

As described above, the polynucleotide of the invention may comprise a transcription regulatory element. Thus, the viral particle of the invention may comprise a transcription regulatory element as described above.

In an embodiment, the viral particle comprises a polynucleotide sequence comprising a transcription regulatory element (comprising e.g. a promoter and/or enhancer), the modified CFI nucleotide sequence, and a poly A sequence, such as the bGHpA sequence. In such embodiments, the poly A sequence, such as the bGHpA sequence, may be located downstream of the modified CFI nucleotide sequence. An intron sequence, such as the SV40 intron sequence, may be located between the transcription regulatory element and the CFI nucleotide sequence.

Compositions, methods and uses

In a further aspect of the invention, there is provided a composition comprising the modified CFI polypeptide, polynucleotide, or vector/viral particle of the invention and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient(s) may comprise carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc. Optionally, the pharmaceutically acceptable excipient(s) comprise saline solution. Optionally, the pharmaceutically acceptable excipient(s) comprises human serum albumin. The invention further provides a modified CFI polypeptide, polynucleotide, vector/viral particle or composition of the invention for use in a method of treatment. Optionally, the method of treatment comprises administering an effective amount of the modified CFI polypeptide, polynucleotide, vector/viral particle, or composition of the invention to a patient.

The invention further provides a method of treatment comprising administering an effective amount of the modified CFI polypeptide, polynucleotide, vector/viral particle, or composition of the invention to a patient.

The invention further provides use of the modified CFI polypeptide, polynucleotide, vector/viral particle or composition of the invention in the manufacture of a medicament for use in a method of treatment. Optionally, the method of treatment comprises administering an effective amount of the modified CFI polypeptide, polynucleotide, vector/viral particle, or composition of the invention to a patient.

Optionally, the method of treatment is a gene therapy. A “gene therapy” involves administering a vector/viral particle of the invention that is capable of expressing a transgene (such as a CFI nucleotide sequence) in the host (e.g. patient) to which it is administered.

Optionally, the method of treatment is a method of treatment by increasing expression of the CFI polypeptide. Optionally, the method of treatment is a method of treatment by increasing activity of the CFI polypeptide.

Optionally, the patient has a lower than normal level of CFI protein (prior to administering the modified CFI polypeptide, polynucleotide, vector/viral particle or composition). Optionally, the patient has a lower than normal level of CFI expression and/or a lower than normal level of CFI activity (e.g. C3b-inactivating and/or iC3b degradation activity) (prior to administering the modified CFI polypeptide, polynucleotide, vector/viral particle or composition). Optionally, the patient has a mutation in the CFI gene or in one or more of the transcription regulatory elements for the CFI gene. Optionally, the patient is deficient in CFI (prior to administering the modified CFI polypeptide, polynucleotide, vector/viral particle or composition). Optionally, the patient has a normal level of CFI protein (prior to administering the modified CFI polypeptide, polynucleotide, vector/viral particle or composition). Optionally, the patient has a normal level of CFI expression and/or a normal level of CFI activity (e.g. C3b-inactivating and/or iC3b degradation activity) (prior to administering the modified CFI polypeptide, polynucleotide, vector/viral particle or composition). Optionally, the patient has a wild-type CFI gene. Optionally, the patient does not have a mutation or defect in an allele of the CFI gene. Optionally, the patient does not have a mutation or defect in one or more of the transcription regulatory elements for the CFI gene. Optionally, the administration of the CFI polypeptide, polynucleotide, vector/viral particle, or composition increases the level of CFI protein. Optionally, the administration of the CFI polypeptide, polynucleotide, viral particle, or composition increases the level of CFI protein above a normal level. Optionally, a normal level of CFI protein is equivalent to that provided by 30-40pg/ml of CFI polypeptide in the serum. Optionally, the increase in the level of CFI protein (e.g. to above a normal level) is an increase in the level of CFI protein in the tissue or organ to which the CFI polypeptide, polynucleotide, vector/viral particle, or composition is administered, such as the eye.

Optionally, the polypeptide is the polypeptide of the invention as described herein. Optionally, the polynucleotide is the polynucleotide of the invention as described herein. Optionally, the viral particle is the viral particle of the invention as described herein. Optionally, the composition is the composition of the invention as described herein.

Optionally, the method of treatment is a method of treating a complement-mediated disorder. Optionally, the complement-mediated disorder is a C3 -mediated disorder. Optionally, the complement-mediated disorder is a kidney disorder. Optionally, the complement-mediated disorder is an ocular disorder. Optionally, the complement- mediated disorder is associated with over-activity of the complement C3b feedback cycle. Optionally, the complement-mediated disorder is selected from C3 glomerulopathy, IgA nephropathy, lupus nephritis, systemic lupus erythematosus, membranous nephropathy, membranoproliferative glomerulonephritis, paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome, autoimmune haemolytic anaemia, ANCA- associated vasculitis, Gaucher disease, peritonitis, age-related macular degeneration, diabetic retinopathy, dense deposit disease, age-related inflammatory or autoinflammatory diseases, autoimmune arthritis such as rheumatoid arthritis, atherosclerosis, chronic cardiovascular disease, Alzheimer’s disease, systemic vasculitis, Guillain-Barre syndrome, and Henoch-Schonlein purpura.

Optionally, the complement-mediated disorder is an age-related inflammatory or autoinflammatory disease. Optionally, the complement-mediated disorder is a chronic inflammatory disease.

Optionally, the complement-mediated disorder is selected from C3 glomerulopathy, C3 glomerulonephritis and dense deposit disease.

Optionally, the complement-mediated disorder is atypical haemolytic uremic syndrome. Optionally, the disorder is atypical haemolytic uremic syndrome with monoallelic CFH mutation. In other words, the complement-mediated disorder may be atypical haemolytic uremic syndrome, and the group of patients to be treated is a subset of atypical haemolytic uremic syndrome patients that have a monoallelic CFH mutation.

Optionally, the complement-mediated disorder is a kidney glomerular or tubular disorder. Optionally, the complement-mediated disorder is selected from C3 glomerulopathy, IgA nephropathy, lupus nephritis and membranous nephropathy. Optionally, the disorder is lupus nephritis. Optionally, the disorder is systemic lupus erythematosus. Optionally, the complement-mediated disorder is atypical hemolytic uremic syndrome (aHUS), stx- associated HUS, cryoglobulinemia, anti-glomerular basement membrane (GBM) disease, ANCA-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated rejection of renal transplant, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

When a disease or disorder is “ treated’’’ as discussed herein (for example in the methods or uses of the invention), this means that one or more symptoms of the disease or disorder are ameliorated. It does not mean that the symptoms of the disease or disorder are completely remedied so that they are no longer present in the patient, although in some methods, this may be the case. Thus, in all instances the term “treatment” or “treating” can be replaced with the term “amelioration" or “ameliorating” , respectively. The methods or uses of the invention (such as the methods of treatment or treating) may result in one or more of the symptoms of the disease or disorder being less severe than before treatment.

In addition, the methods or uses of the invention may “prevent” disorders. In some embodiments, the term “treatment” or “treating” can be replaced with the term “prevention” or “preventing” , respectively.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An example of a desired therapeutic result is raising the level of functional CFI in a subject (e.g. so as to lead to functional CFI production at a level sufficient to ameliorate the symptoms of the disease or disorder).

Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered intravenously, systemically, to the liver via peripheral vein infusion, to the liver via hepatic vessels such as hepatic vein infusion or hepatic artery infusion, or via intraparenchymal administration direct to the liver. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered intravenously.

Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered systemically. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered to the liver via peripheral vein infusion.

Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered to the liver via hepatic vessels such as hepatic vein infusion or hepatic artery infusion. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered via intraparenchymal administration direct to the liver.

Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by injection into the renal artery. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by retrograde administration, e.g. via the ureters using a urinary catheter. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered via direct kidney parenchymal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered via sub-capsular injection.

Optionally, the method of treatment is a method of treating an ocular disorder. Optionally, the method of treatment is a method of treating a complement-mediated disorder.

Optionally, the complement-mediated disorder is an ocular disorder. Optionally, the ocular disorder is a chronic complement-mediated inflammatory condition of the eye. Optionally, the ocular disorder is age-related macular degeneration (AMD) or diabetic retinopathy. Optionally, the ocular disorder is glaucoma, Stargardt’s disease, central serous chorioretinopathy or retinitis pigmentosa. Optionally, the ocular disorder is diabetic macular edema (DME), keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, uveitis, or vitreoretinopathy. Optionally, the ocular disorder is non-oncological. Optionally, the ocular disorder is AMD. Optionally, the AMD is dry AMD. Optionally, the AMD is geographic atrophy (GA).

Optionally, the method of treatment is a method of treating AMD. Optionally, the method of treatment is a method of treating dry AMD. Optionally, the method of treatment is a method of treating GA.

Optionally, the method of treatment reduces the rate of, or prevents, the progression of a degenerative-type disorder, such as AMD. The clinical progression of AMD is characterised in stages according to changes in the macula. The hallmark of early AMD is drusen, which are accumulations of extracellular debris underneath the retina and appear as yellow spots in the retina on clinical exam and on fundus photographs. Drusens are categorised by size as small (<63pm), medium (63-124 pm) and large (>124pm). They are also considered as hard or soft depending on the appearance of their margins on opthalmological examination. Hard drusens have clearly defined margins. Soft ones have less defined and fluid margins. The Age-Related Eye Disease Study (AREDS) fundus photographic severity scale is one of the main classification systems used for this condition. Dry AMD is typically characterized by progressive apoptosis of cells in the retinal pigment epithelium (RPE) layer, the overlying photoreceptor cells, and frequently also the underlying cells in the choroidal capillary layer. Confluent areas of RPE cell death accompanied by overlying photoreceptor atrophy are referred to as geographic atrophy (GA). Patients with this form of AMD experience a slow and progressive deterioration in central vision.

Optionally, the method of treatment results in a prevention of, or reduction in, the rate of appearance of one or more AMD characteristics as described above. Optionally, the method of treatment results in the maintenance, or improvement in, visual function in a patient with AMD.

Optionally, the method of treatment results in the treatment of GA. Optionally, the method of treatment results in a prevention of, or reduction in, GA. Optionally, the method of treatment results in a prevention of an increase in the area of GA. Optionally, the method of treatment results in a reduction of the amount of GA. Optionally, the method of treatment results in a reduction in the area of GA. Optionally, the method of treatment results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in the amount of GA (e.g. the area of GA) at least 12 months (e.g. at 12 months) from the administration (which is the first administration in the case of multiple administrations) of the CFI polypeptide, polynucleotide, vector/viral particle and/or composition to an eye. Optionally, the method of treatment results in a prevention of, or reduction in, the formation of GA. Optionally, the method of treatment results in a prevention of, or reduction in the rate of, progression of GA. Optionally, the method of treatment results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in the rate of the increase in GA area at least 12 months (e.g. at 12 months) from the administration (which is the first administration in the case of multiple administrations) of the CFI polypeptide, polynucleotide, vector/viral particle and/or composition to an eye, relative to an untreated eye over the same period.

Optionally, the method of treatment results in a prevention of, or reduction in the rate of, formation of drusen. Optionally, the method of treatment results in a reduction in existing drusen, for example a reduction in the size and/or number of existing drusen. Optionally, the method of treatment results in a prevention of, or reduction in the rate of, complement deposition. Optionally, the method of treatment results in a reduction in existing complement deposition.

Optionally, the method of treatment results in an improvement in, or restoration of, vision or visual acuity. Optionally, the method of treatment mitigates the loss of vision or visual acuity. Optionally, the method of treatment results in an improvement in, or restoration of, reading speed in a patient. Optionally, the method of treatment mitigates the reduction in reading speed in a patient. Optionally, the method of treatment results in a reduction in, or prevention of, loss of photoreceptors and/or the retinal pigment epithelium (RPE).

Optionally, the ocular disorder is AMD. Optionally, the method of treatment is a method of treating AMD. Optionally, the AMD is dry AMD. Optionally, the method of treatment is a method of treating dry AMD. Optionally, the AMD is GA. Optionally, the method of treatment is a method of treating GA.

Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by subretinal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by direct retinal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by suprachoroidal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered by intravitreal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is administered intraocularly.

Optionally, the method of treatment is a method of treating a non-ocular disorder. Optionally, the method of treatment is a method of treating a complement-mediated disorder. Optionally, the complement-mediated disorder is a non-ocular disorder. Optionally, the non-ocular disorder is a systemic acute disorder. Optionally, the non- ocular condition is a systemic acute disorder selected from the group consisting of: acute glomerulonephritis, acute renal injury, acute respiratory distress syndrome, bacterial meningitis, brain haemorrhage, bums, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia reperfusion injury, Lyme disease, myocardial infarction, organ transplantation, periodontitis, pneumonia, pre-eclampsia, schistosomiasis, sepsis, stroke, thromboembolism, ischemia-reperfusion injury and traumatic brain injury. Optionally, the non-ocular disorder is a systemic chronic disorder. Optionally, the non-ocular disorder is a systemic chronic disorder selected from the group consisting of: Alzheimer’s disease, anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic kidney failure, chronic obstructive pulmonary disease, Crohn’s disease, diabetic neuropathy, generalized myasthenia gravis (gMG), Granulomatosis with Polyangiitis (GPA), Guillain-Barre Syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis (MS), non-insulin dependent diabetes, osteoarthritis, pancreatitis, Parkinson’s disease, paroxysmal nocturnal hemoglobinuria, post-transplant lymphoproliferative disease, protein losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), ulcerative colitis, Amyotrophic lateral sclerosis (ALS), warm autoimmune hemolytic anemia (wAIHA), cold agglutinin disease (CAD), Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN), Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA), Huntington’s disease and chronic inflammatory demyelinating polyneuropathy (CIDP).

Optionally, the disorder is not an ocular disorder. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle, or composition is not administered intraocularly.

Optionally, the CFI polypeptide, the polynucleotide, vector/viral particle, or composition is not administered by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection. Optionally, the CFI polypeptide, the polynucleotide, vector/viral particle, or composition is not administered by subretinal injection.

Optionally, the CFI polypeptide, the polynucleotide, vector/viral particle, or composition is not administered by direct retinal injection. Optionally, the CFI polypeptide, the polynucleotide, vector/viral particle, or composition is not administered by suprachoroidal injection. Optionally, the CFI polypeptide, the polynucleotide, vector/viral particle, or composition is not administered by intravitreal injection. Optionally, the CFI polypeptide, polynucleotide, vector/viral particle and/or composition is not administered intraocularly.

Optionally, administration of the CFI polypeptide, polynucleotide, vector/viral particle, or composition increases the level of C3b-inactivating and/or iC3b-degradation activity in the patient. Optionally, the administration of the CFI polypeptide, the polynucleotide, vector/viral particle or composition increases the level of C3b-inactivating and/or iC3b- degradation activity to a normal level. Optionally, administration of the CFI polypeptide, the polynucleotide, vector/viral particle or composition increases the level of C3b- inactivating and/or iC3b-degradation activity above a normal level. Optionally, administration of the CFI polypeptide, the polynucleotide, vector/viral particle or composition increases the level of C3b-inactivating and/or iC3b-degradation activity to a level 1.2-fold, 1.5-fold, 1.8-fold or 2-fold higher than the normal level. Optionally, a normal level is equivalent to that provided by 30-40pg/ml of CFI polypeptide in the serum. Optionally, the increase in the level of C3b-inactivating and/or iC 3b -degradation activity is an increase in the level of C3b-inactivating and/or iC3b-degradation activity (e.g. to above a normal level) in the tissue or organ to which the CFI polypeptide, polynucleotide, vector/viral particle, or composition is administered, such as the eye. For example, administration of the CFI polypeptide, polynucleotide, vector/viral particle or composition by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection may increase the level of C3b-inactivating and/or iC3b-degradation activity (e.g. to above a normal level) in the eye.

Optionally, the method of treatment is a method for increasing the level of C3b- inactivating and/or iC3b-degradation activity in a patient. Optionally, the method of treatment is a method for increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient, thereby treating the complement-mediated disorder. Optionally, the method of treatment is a method for treating a disease by increasing the level of C3b- inactivating and/or iC3b-degradation activity in a patient. Optionally, the method of treatment is a method for treating a disorder by increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient. Optionally, increasing the level of C3b- inactivating and/or iC3b-degradation activity in a patient is increasing the level of C3b- inactivating and/or iC3b-degradation activity to a normal level. Optionally, increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient is increasing the level of C3b-inactivating and/or iC3b-degradation activity above a normal level.

Optionally, increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient is increasing the level of C3b-inactivating and/or iC3b-degradation activity to a level 1.2-fold, 1.5-fold, 1.8-fold or 2-fold higher than the normal level. Optionally, a normal level is equivalent to that provided by 30-40pg/ml of CFI polypeptide in the serum. Optionally, the increase in the level of C3b-inactivating and/or iC 3b -degradation activity is an increase in the level of C3b-inactivating and/or iC3b-degradation activity (e.g. to above a normal level) in the tissue or organ to which the CFI polypeptide, polynucleotide, vector/viral particle, or composition is administered, such as the eye. For example, administration of the CFI polypeptide, polynucleotide, vector/viral particle or composition by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection may increase the level of C3b-inactivating and/or iC3b-degradation activity (e.g. to above a normal level) in the eye.

The invention will now be described with reference to the following examples, which are merely illustrative and should not in any way be construed as limiting the scope of the present invention.

EXAMPLES

Example 1 - GENERAL METHODS

Unless specified otherwise, the following general methods were followed in the examples described below.

Generation of CFI proteins

N-terminal 6His-tagged CFI variants or wild-type CFI were expressed using Expi293F cells (Thermo Fisher, Cat. no. A14527). These CFI variants correspond to wild-type CFI except they contain certain amino acid substitutions.

Preparation of Expi293F cells for transfection

Expi293F cells were expanded in suspension in 30 mL Expi293 medium (Thermo Fisher, Cat. no. A1435101) at 37°C, 125 rpm, 8% CO2 atmosphere with 80% humidity in 125 mL plastic flasks with ventilated caps (Thermo Scientific, Cat. no. 4115-0125). During the maintenance and expansion phase, cells were passaged to 0.3 xlO⁶ viable cells per mL when they reached a density of 3-5xl0⁶ cells/mL. Cell viability was determined using an automated cell counter (Thermo Fisher, Cat. No. AMQAX2000) and trypan blue exclusion (Thermo Fisher, Cat No. T10282).

During each passage, cells were spun down at 400 x g for 10 minutes at room temperature in 50 mL centrifuge tubes (Corning, Cat. No. 430829) and media was removed by aspiration before resuspending the cells in pre-warmed fresh Expi293 medium.

Preparation of the transgene-expressing plasmids

Plasmids for expressing the proteins (e.g. a CFI variant or wild-type CFI) were manufactured by GenScript™. Each plasmid contained a codon-optimised transgene encoding a CFI variant and a native signal peptide (e.g. SEQ ID NO: 17 for PL135 in Table 2 below) or wild-type CFI and a native signal peptide (SEQ ID NO: 16). Each transgene comprised a sequence encoding a 6His-tag between the sequence encoding the signal peptide and the sequence encoding the mature CFI sequence. The plasmids were lyophilized for storage.

Preparation of the transfection complex

A total of 40 pL of nuclease-free water (Invitrogen, Cat. No. AM9937) was added to 4 pg of lyophilized plasmid DNA containing a CFI variant or wild-type CFI transgene (prepared according to the section entitled “ Preparation of the transgene-expressing plasmids” above). A total of 7 pL of each reconstituted plasmid DNA was transferred to a 96-well plate (V -bottom shaped; Coming, REF 3894). A total of 3.5 mL OptiMEM (Gibco) was mixed with 70 pL of a Ipg/pl solution of pCMV6-AC-GFP vector containing a transgene encoding human Furin (Origene Technologies Biotechnology, CAT. No. RG204279-FURIN) to form the OptiMEM-Furin mix. 70 pL of the OptiMEM-Furin mix was then added to each well of the V-bottom shaped 96-well plate containing the reconstituted plasmid DNA and incubated for 5 minutes. A working solution of ExpiFectamine-OptiMEM was made by adding 400 uL ExpiFectamine (Thermo Fisher, Cat. No. A14524) to 7.3 mL OptiMEM in a 15 mL centrifuge tube (Corning, CAT. No. 430766). 74 pL of the working solution of ExpiFectamine-OptiMEM was added to each well of the V-bottom shaped 96-well plate (already containing the reconstituted plasmid DNA and OptiMEM-Furin mix) and incubated at room temperature. After a 20-30-minute incubation, the resulting mixture (containing DNA-lipid complexes) was ready to be used for transfection.

Transfection of Expi293F cells

On the day of transfection, Expi293F cells (prepared in accordance with the section above entitled “ Preparation of Expi293F cells for transfection”) WCK adjusted to a density of 2.8 x 10⁶ cells/mL using pre-warmed Expi293 medium, and 0.7 mL of the cells in media were added per well of 96-deep-well plates (Fisher Scientific, Cat No. 10447181). Each plate was sealed with a porous tape (Invitrogen, Cat No. 10544453) and placed in a microplate rack in the shaker incubator at 400 rpm until the DNA-lipid complexes (as prepared in accordance with the section above entitled “ Preparation of the transfection complex”) were ready.

70 pL of the mixture containing DNA-lipid complexes (prepared in accordance with the section above entitled “Preparation of the transfection complex”) was added to each well containing the Expi293F cells. The plate was re-sealed, and the cells were cultured at 37 °C, in a 8% CO2 humidified atmosphere with shaking (400 rpm) for 18 to 24 hours.

Following the initial incubation for 18 to 24 hours, a cocktail of Enhancer 1 and Enhancer 2 from the ExpiFectamine 293 transfection kit (Thermo Fisher, Cat. No. A14524) was added (27.5 pL/well). The plate was then re-sealed and returned to the humidified 8% CO2 incubator at 37°C shaking at 400 rpm for a further four days.

Supernatants were harvested by centrifugation of the plate at 400 x g for 10 minutes. The supernatants were transferred to a new 96-deep well plate and centrifuged at 2000 x g for 10 minutes. The supernatants were filtered with 3.0 pm/0.2 pm vacuum filters (PALL CAT. No. 5053) and stored in a new 96-deep well plate at 4°C until analysis. For example, some supernatants were used in the “Quantification of Human CFI protein using ELISA ” or “ CFI activity determination through measurement of iC3b formation using an HTRF assay” as set out below.

Quantification of Human CFI protein using ELISA

The concentration of CFI protein expressed and secreted from Expi293F cells was quantified using a Complement Factor I Human ELISA kit (Hycult, CAT. No. HK355-01). The ELISA was performed according to assay instructions. In brief, supernatant samples (diluted with the sample diluent from the kit as necessary) and human CFI standards from the kit were incubated in microtiter wells coated with anti-human CFI antibodies (to capture the human CFI). A biotinylated tracer antibody was added (to bind to the captured human CFI) followed by a streptavidin-peroxidase conjugate (to bind to the biotinylated tracer antibody). The substrate tetramethylbenzidine (TMB) was then added, which the streptavidin-peroxidase conjugate reacts with. The enzyme reaction was stopped by adding oxalic acid. For each of the samples and standards, the absorbance at 450 nm was measured with a spectrophotometer. A standard curve was obtained by plotting the absorbance (linear) against the corresponding concentrations of the human CFI standards (log). The concentration of human CFI in the samples was then determined from the standard curve.

CFI activity determination through measurement of iC3b formation using an HTRF assay

The activity of CFI variants and wild-type CFI was evaluated by measuring CFI cleavage of C3b into iC3b using an homogeneous time resolved fluorescence (HTRF) assay. Cleavage of C3b was measured using a monoclonal IgG antibody specific for human iC3b neoantigen conjugated to terbium cryptate (CisBio, Order #64CUSTAYE, '''anli-iC3h (Tb) IgG”, ) and an anti-C3b/iC3b antibody conjugated to fluorescein (FITC) (CEDARLANE CAT. No. CL7632F, “anti-C3b (FITC) IgG”). Supernatants (obtained from the transfection of Expi293F cells as described above) containing CFI protein (e.g. a CFI variant or wild-type CFI) were diluted in 50 mM HEPES (Merck, CAT. NO. H3375), 0.1% BSA (Merck, CAT. NO. A4503), 0.1% Tween- 20 (Acres Organics, CAT. NO. 233362500), pH 7.0 so the CFI protein concentration was, for example, 150 pM (for CFI variants comprising single amino acid mutations) or 75 pM (for CFI variants comprising double, triple, quadruple, or quintuple amino acid mutations). The supernatant was diluted by a minimum of 1 : 100. The diluted supernatants containing CFI protein were added to white, low-volume 384-well plates (Sigma, CAT. No. 784904) at a volume of 5 pL per well. 5 pL of a master mix containing 9 nM C3b (Complement Technology, CAT. NO. Al 14) and 6 nM cofactor H (CFH) (Complement Technology, CAT. NO. A137) was added to each well using an automated dispenser (MANTIS, FORMULATRIX®). The reaction was incubated for, for example, 1 hour (for CFI variants comprising single amino acid mutations) or 30 minutes (for CFI variants comprising double, triple, quadruple, or quintuple amino acid mutations) at room temperature. For the time-response curve experiment (Figure 2), the reaction was incubated for 1 hour, 2 hours, 3 hours or 5 hours. 5 pL of an antibody master mix containing anti-iC3b (Tb) IgG at 1.5 pg/mL and anti-C3b (FITC) IgG at 36 nM was then added to each well. The reaction was incubated for a further, for example, 1 hour (for CFI variants comprising single amino acid mutations) or 30 minutes (for CFI variants comprising double, triple, quadruple, or quintuple amino acid mutations) at room temperature. For the time-response curve experiment (Figure 2), the reaction was incubated for a further 1 hour. The volume of the reaction following the addition of the antibody master mix was 15 pL and the concentration of CFI protein was, for example, 50 pM (for CFI variants comprising single amino acid mutations) or 25 pM (for CFI variants comprising double amino acid mutations). Each reaction was run in triplicate.

Calculating HTRF Ratio

CFI activity was assessed fluorometrically using Fluorescence Resonance Energy Transfer (FRET) technology. The reaction mixture (from the above method following the incubation with the antibody master mix) was exposed to an excitation wavelength of 340 nm (Xex= 340 nm) using a SpectraMax® i3 plate reader (Molecular Devices). Fluorescent readings were taken (using a SpectraMax® i3 plate reader (Molecular Devices)) at 490 nM for the emission spectra of terbium cryptate and at 520 nm for the emission spectra of FITC (lem= 490 and 520 nm). Readings were taken from a height of 3 mm above the plate and with an integration time of 0.5 ms. The mean fluorescence intensity (MFI) was calculated for both the 490 nm and 520 nm channels for each reaction. The HTRF ratio was then calculated by dividing the MFI measured at 520nm (HTRF FRET acceptor) by the MFI measured at 490 nm (HTRF FRET donor), and multiplying the resulting value by 10,000 (as shown below):

HTRF ratio

Calculating the CFI activity relative to a wild type CFI

Wild-type CFI was used to standardise and compare the activity of the mutations across some experiments. For some experiments, the HTRF ratio for a CFI variant was divided by the HTRF ratio for wild type CFI in order to obtain the fold change of CFI activity relative to wild type CFI.

Calculating percentage conversion of C3b to iC3b (% of iC3b formation)

For the time-response curve experiment (Figure 2), the percentage of C3b conversion to iC3b (i.e. the % of iC3b formation) was calculated using an iC3b standard curve. To generate the standard curve, reaction mixtures containing different ratios of C3b to iC3b, but with an equal total protein concentration, were prepared in assay buffer (50 mM HEPES, pH 7.0, supplemented with 0.1% BSA and 0.1% Tween-20) in the absence of any CFI.

The reaction mixtures were incubated for 1, 2, 3, or 5 hours (i.e. the same lengths of time that C3b and CFH were incubated with the corresponding CFI variants or wild type CFI). The antibody master mix was added to the reaction mixtures and the reactions were incubated for a further 1 hour. The total concentration of iC3b plus C3b once the antibody master mix was added was 3 nM. Table 1 shows the concentrations of iC3b and C3b in the reaction mixtures following the addition of the antibody master mix.

Table 1: HTRF assay C3b and iC3b concentrations in the reaction mixtures following the addition of the antibody master mix. Each ratio of C3b to iC3b represents the ratio that would be present for a particular percentage of iC3b formation from C3b. The reaction mixtures were used to generate the iC3b standard curve.

The HTRF ratio for each reaction mixture was then calculated using the method described above. A standard curve of the HTRF ratio against percentage iC3b formation was then plotted. The HTRF ratio for each of the CFI variants and wild type CFI were then fitted by interpolation against the standard curve to calculate the percentage conversion of C3b to iC3b.

Statistical Analysis

Data were analysed and graphed using GraphPad Prism 7.0.3 or 8.0.2 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± SD. CFI time response curves in Figure 2 were calculated by non-linear regression (four variable slope).

Example 2 - CFI variants

Table 2: List of CFI variants

The variants in Table 2 all have a sequence of SEQ ID NO: 16 except for the respective listed mutation(s) (other than PL01). The positions of the mutations indicated in Table 2 are the corresponding positions in the immature CFI polypeptide sequence (i.e. the corresponding positions in the polypeptide sequence of SEQ ID NO: 1). The nucleotide sequence of SEQ ID NO: 16 comprises a sequence encoding a 6His-tag between the sequence encoding the signal peptide and the sequence encoding the mature CFI sequence. Thus, the positions of the amino acids of the mature CFI polypeptide in the sequence of SEQ ID NO: 1 and the corresponding positions of the codons encoding said amino acids of the mature CFI polypeptide in SEQ ID NO: 16 are transposed by six. For example, position 53 in SEQ ID NO: 1 corresponds to position 59 (i.e. 53 plus 6) in the sequence encoded by SEQ ID NO: 16. In other words, position 53 in SEQ ID NO: 1 corresponds to the codon in SEQ ID NO: 16 encoding position 59 (i.e. 53 plus 6). Therefore, the variants in Table 2 all have a sequence of SEQ ID NO: 16 except that (other than PL01) the codon(s) in SEQ ID NO: 16 corresponding to the respective indicated position(s) in Table 2 (i.e. the corresponding position(s) in SEQ ID NO: 1) encode the respective substituted amino acid(s).

For example, PL53 (I409F) has a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 409 in SEQ ID NO: 1 encodes phenylalanine. Thus, PL53 (I409F) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 415 (i.e. 409 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes phenylalanine. In a further example, PL133 (R53L/R383I) has a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 53 in SEQ ID NO: 1 encodes leucine and the codon which corresponds to position 383 in SEQ ID NO: 1 encodes isoleucine. Thus, PL133 (R53L/R383I) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 59 (i.e. 53 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes leucine and the codon which encodes position 389 (i.e. 383 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes isoleucine. In yet another example, PL211 (R383E/G439Q/I409F) has a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 383 in SEQ ID NO: 1 encodes glutamic acid, the codon which corresponds to position 439 in SEQ ID NO: 1 encodes glutamine and the codon which corresponds to position 409 in SEQ ID NO: 1 encodes phenylalanine. Thus, PL211 (R383E/G439Q/I409F) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 389 (i.e. 383 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes glutamic acid, the codon which encodes position 445 (i.e. 439 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes glutamine and the codon which encodes position 415 (i.e. 409 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes phenylalanine. In another example, PL366 (R53Y/R383Y/V408T/H401Y) has a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 53 encodes tyrosine, the codon which corresponds to position 383 encodes tyrosine, the codon which corresponds to position 408 encodes threonine and the codon which corresponds to position 401 encodes tyrosine. Thus, PL366 (R53Y/R383Y/V408T/H401Y) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 59 (i.e. 53 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes tyrosine, the codon which encodes position 389 (i.e. 383 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes tyrosine, the codon which encodes position 414 (i.e. 408 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes threonine, and the codon which encodes position 407 (i.e. 401 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes tyrosine. In a further example, PL102 (R383LH401Y/I409F/E479D/F482Q) has a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 383 encodes isoleucine, the codon which corresponds to position 401 encodes tyrosine, the codon which corresponds to position 409 encodes phenylalanine, the codon which corresponds to position 479 encodes aspartic acid and the codon which corresponds to position 482 encodes glutamine. Thus, PL 102 (R383LH401Y/I409F/E479D/F482Q) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 389 (i.e. 383 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes isoleucine, the codon which encodes position 407 (i.e. 401 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes tyrosine, the codon which encodes position 415 (i.e. 409 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes phenylalanine, the codon which encodes position 485 (i.e. 479 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes aspartic acid and the codon which encodes position 488 (i.e. 482 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes glutamine. PL135 (R53L/R383Y) can have a sequence of SEQ ID NO: 17 as indicated in the table, or it can have a sequence of SEQ ID NO: 16 except that the codon which corresponds to position 53 in SEQ ID NO: 1 encodes leucine and the codon which corresponds to position 383 in SEQ ID NO: 1 encodes tyrosine. Thus, PL135 (R53L/R383Y) has a sequence of SEQ ID NO: 16 except that the codon which encodes position 59 (i.e. 53 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes leucine and the codon which encodes position 389 (i.e. 383 plus 6) in the sequence encoded by SEQ ID NO: 16 encodes tyrosine.

Numerous CFI variants were generated. These CFI variants correspond to wild-type CFI except they contain specific amino acid substitutions such as those shown in Table 2 (as well as a 6His-tag between the signal peptide and mature CFI polypeptide). Standard procedures can be used to generate CFI variants, such as site-directed mutagenesis. Plasmids for expressing the proteins were generated according to the section entitled "''Preparation of the transgene-expressing plasmids” in Example 1.

The CFI variants were tested for their iC3b formation activity. In short, the CFI variants were expressed and secreted according to the methods described in the sections entitled “Generation of CFI proteins”, “Preparation of Expi293F cells for transfection” , “Preparation of the transgene-expressing plasmids”, “Preparation of the transfection complex” and “Transfection of Expi293F cells” in Example 1. The concentration of CFI protein expressed and secreted from the Expi293F cells was quantified according to the section entitled “Quantification of Human CFI protein using ELISA ” . The CFI iC3b formation activity of the CFI protein expressed and secreted from the Expi293F cells was measured according to the methods described in the section entitled “CFI activity determination through measurement of iC3b formation using an HTRF assay”.

Numerous CFI variants had a higher CFI iC3b formation activity than wild-type CFI. The results are discussed further in the Examples below.

Example 3 - Increased CFI iC3b formation activity achieved by CFI variants comprising single amino acid mutations Numerous CFI variants comprising single amino acid mutations were generated, along with wild-type (WT) CFI (PL01). For determining the CFI iC3b formation activity, the supernatants containing the CFI variants were diluted so that the CFI variant protein concentration was 150 pM (50 pM following the addition of the antibody master mix). After the 1 hour incubation with the C3b/CFH master mix and the 1 hour incubation with the antibody master mix, the HTRF ratio was calculated for each CFI variant and wild-type CFI . The HTRF ratio for each CFI variant was divided by the HTRF ratio of the wildtype CFI in order to obtain the fold change in activity for each CFI variant relative to wild type CFI. Numerous variants had activity as good as or higher than that of wild-type CFI (see Figure 1 which shows a subset of these variants). A negative control (PL10) was also included and showed no iC3b formation activity (data not shown). “100% iC3b” in Figure 1 is a control showing the HTRF ratio (plotted as fold-change relative to the HTRF ratio of wild-type CFI) achieved for 3nM of iC3b in the absence of any CFI. Thus, iC3b was incubated in the same manner as the CFI variants and wild type CFI, except in the absence of any CFI and C3b, and the concentration of iC3b following the addition of the antibody master mix was 3 nM.

The iC3b formation activity of some of the highest CFI variants comprising single amino acid mutations was measured using the CFI HTRF activity assay following multiple incubation times with C3b and CFH, and the antibodies (2, 3, 4 and 6 hours). The percentage of iC3b formed (% of iC3b formation) was calculated according to the section entitled “ CFI activity determination through measurement of iC3b formation using an HTRF assay” and the results are shown in Figure 2.

Example 4 - CFI iC3b formation activity achieved by CFI variants comprising double amino acid mutations

Various single CFI variants were selected, based on their gain of activity, their relative location at the different interfaces, as well as the nature of their amino acid substitutions, for the design of the double CFI variants. Numerous CFI variants comprising double amino acid mutations were analysed and compared with wild-type CFI. To avoid the CFI iC3b formation activity assay from reaching saturation (i.e. to avoid all the C3b from being converted to iC3b during the assay) and therefore enable a better comparison between the CFI variants and wild-type CFI, the CFI variants comprising double amino acid mutations were diluted to 75 pM (25 pM in the reaction following the addition of the antibody master mix). After the 30 minute incubation with the C3b/CFH master mix and the 30 minute incubation with the antibody master mix, the HTRF ratio was calculated for each CFI variant and wild-type CFI. The HTRF ratio for each CFI variant was divided by the HTRF ratio of the wild-type CFI in order to obtain the fold change in activity for each CFI variant relative to wild type CFI. A number of CFI variants showed similar or increased iC3b formation compared to wildtype CFI (see Figure 3 which shows a subset of these variants). “100% iC3b” in Figure 3 is a control showing the HTRF ratio (plotted as fold-change relative to the HTRF ratio of wild-type CFI) achieved for 3nM of iC3b in the absence of any CFI. Thus, iC3b was incubated in the same manner as the CFI variants and wild type CFI, except in the absence of any CFI and C3b, and the concentration of iC3b following the addition of the antibody master mix was 3 nM.

Example 5 - CFI iC3b formation activity achieved by CFI variants comprising triple, quadruple or quintuple amino acid mutations

Numerous CFI variants comprising triple, quadruple or quintuple mutations were analysed and compared with wild-type CFI. To avoid the CFI iC3b formation activity assay from reaching saturation (i.e. to avoid all the C3b from being converted to iC3b during the assay) and therefore to enable a better comparison between the CFI variants and wild-type CFI, the CFI variants comprising the triple, quadruple, or quintuple amino acid mutations were diluted to 75 pM (25 pM in the reaction following the addition of the antibody master mix). After the 30 minute incubation with the C3b/CFH master mix and the 30 minute incubation with the antibody master mix, the HTRF ratio was calculated for each CFI variant and wild-type CFI. The HTRF ratio for each CFI variant was divided by the HTRF ratio of the wild-type CFI in order to obtain the fold change in activity for each CFI variant relative to wild type CFI.

A number of CFI variants showed similar or increased iC3b formation compared to wildtype CFI (see Figure 7A and B which show a subset of these variants). “100% iC3b” in Figure 7 A and B is a control showing the HTRF ratio (plotted as fold-change relative to the HTRF ratio of wild-type CFI) achieved for 3nM of iC3b in the absence of any CFI. Thus, iC3b was incubated in the same manner as the CFI variants and wild type CFI, except in the absence of any CFI and C3b, and the concentration of iC3b following the addition of the antibody master mix was 3 nM.

Aspects of the Invention

1. A modified complement factor I (CFI) polypeptide comprising one or more mutations, wherein the one or more mutations provides higher CFI activity.

2. A modified CFI polypeptide comprising one or more mutations, wherein the one or more mutations comprises a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

3. The modified CFI polypeptide of aspect 1 or 2, wherein the one or more mutations comprises:

(i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b; and/or

(ii) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H.

4. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more or two or more positions within the modified CFI polypeptide which can form an interface with C3b.

5. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions within the modified CFI polypeptide which can form an interface with Factor H.

6. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with C3b; and (ii) a mutation at one or more positions within the modified CFI polypeptide which can form an interface with Factor H.

7. The modified CFI polypeptide of aspect 4 or 6, wherein at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the SP domain.

8. The modified CFI polypeptide of any one of aspects 4 and 6 to 7, wherein at least one of the positions within the modified CFI polypeptide which can form an interface with C3b is in the FIM domain.

9. The modified CFI polypeptide of any one of aspects 4 and 6 to 8, wherein the interface with C3b is the interface with the C3b alpha chain.

10. The modified CFI polypeptide of any one of aspects 4 and 6 to 9, wherein the interface with C3b is the interface with the CUB domain in the C3b alpha chain.

11. The modified CFI polypeptide of any one of aspects 4 and 6 to 10, wherein the interface with C3b is the interface with the substrate loop within the CUB domain in the C3b alpha chain.

12. The modified CFI polypeptide of any one of aspects 4 and 6 to 9, wherein the interface with C3b is the interface with the CTC domain in the C3b alpha chain.

13. The modified CFI polypeptide of any one of aspects 4 and 6 to 8, wherein the interface with C3b is the interface with the C3b beta chain.

14. The modified CFI polypeptide of aspect 5 or 6, wherein at least one of the positions within the modified CFI polypeptide which can form an interface with Factor H is in the SP domain.

15. The modified CFI polypeptide any one of aspects 5, 6 or 14, wherein the interface with Factor H is the interface with the CCP2 domain in Factor H.

16. The modified CFI polypeptide of any one of aspects 5, 6 and 14 to 15, wherein the interface with Factor H is the interface with the CCP3 domain in Factor H. 17. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

18. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 60, 383, 384, 401, 407, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1.

19. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 408, 409, 439, 441, 479, and 482 of SEQ ID NO: 1.

20. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at one or more or two or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, 441, and 479 of SEQ ID NO: 1.

21. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at two or more positions corresponding to a position selected from the group consisting of 53, 383, 401, 409, 439, and 479 of SEQ ID NO: 1.

22. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation to a more hydrophobic amino acid.

23. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1. 24. The modified CFI polypeptide of aspect 23, wherein the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine, or glutamic acid.

25. The modified CFI polypeptide of aspect 23, wherein the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R53I.

26. The modified CFI polypeptide of aspect 23, wherein the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L.

27. The modified CFI polypeptide of aspect 23, wherein the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y.

28. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 60 of SEQ ID NO: 1.

29. The modified CFI polypeptide of aspect 28, wherein the mutation at a position corresponding to position 60 of SEQ ID NO: 1 is a substitution with serine or glutamic acid.

30. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1.

31. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine, glutamic acid, or phenylalanine.

32. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R383I. 33. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with leucine, optionally R383L.

34. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R383Y.

35. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally R383E.

36. The modified CFI polypeptide of aspect 30, wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally R383F.

37. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 384 of SEQ ID NO: 1.

38. The modified CFI polypeptide of aspect 37, wherein the mutation at a position corresponding to position 384 of SEQ ID NO: 1 is a substitution with isoleucine, tryptophan, tyrosine, or phenylalanine.

39. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 400 of SEQ ID NO: 1.

40. The modified CFI polypeptide of aspect 39, wherein the mutation at a position corresponding to position 400 of SEQ ID NO: 1 is a substitution with tryptophan, tyrosine, or arginine.

41. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 401 of SEQ ID NO: 1. 42. The modified CFI polypeptide of aspect 41, wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with glycine, tyrosine, phenylalanine, or tryptophan.

43. The modified CFI polypeptide of aspect 41, wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with glycine, optionally H401G.

44. The modified CFI polypeptide of aspect 41, wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally H401Y.

45. The modified CFI polypeptide of aspect 41, wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally H401F.

46. The modified CFI polypeptide of aspect 41, wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tryptophan, optionally H401W.

47. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 407 of SEQ ID NO: 1.

48. The modified CFI polypeptide of aspect 47, wherein the mutation at a position corresponding to position 407 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I407F.

49. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 408 of SEQ ID NO: 1.

50. The modified CFI polypeptide of aspect 49, wherein the mutation at a position corresponding to position 408 of SEQ ID NO: 1 is a substitution with threonine or tyrosine. 51. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 409 of SEQ ID NO: 1.

52. The modified CFI polypeptide of aspect 51 , wherein the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, tryptophan, or glutamic acid.

53. The modified CFI polypeptide of aspect 51 , wherein the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I409F.

54. The modified CFI polypeptide of aspect 51 , wherein the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with tryptophan, optionally I409W.

55. The modified CFI polypeptide of aspect 51 , wherein the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally I409E.

56. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 439 of SEQ ID NO: 1.

57. The modified CFI polypeptide of aspect 56, wherein the mutation at a position corresponding to position 439 of SEQ ID NO:1 is a substitution with leucine, glutamic acid or glutamine.

58. The modified CFI polypeptide of aspect 56, wherein the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally G439E.

59. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 440 of SEQ ID NO: 1. 60. The modified CFI polypeptide of aspect 59, wherein the mutation at a position corresponding to position 440 of SEQ ID NO: 1 is a substitution with threonine, optionally N440T.

61. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 441 of SEQ ID NO: 1.

62. The modified CFI polypeptide of aspect 61, wherein the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with glutamic acid, phenylalanine or tyrosine.

63. The modified CFI polypeptide of aspect 61, wherein the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally K441E.

64. The modified CFI polypeptide of aspect 61, wherein the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally K441F.

65. The modified CFI polypeptide of aspect 61, wherein the mutation at a position corresponding to position 441 of SEQ ID NO: 1 is a substitution with tyrosine, optionally K441Y.

66. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 479 of SEQ ID NO: 1.

67. The modified CFI polypeptide of aspect 66, wherein the mutation at a position corresponding to position 479 of SEQ ID NO: 1 is a substitution with aspartic acid, optionally E479D.

68. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises a mutation at a position corresponding to position 482 of SEQ ID NO: 1. 69. The modified CFI polypeptide of aspect 68, wherein the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with tryptophan, leucine, histidine, isoleucine, glutamine, arginine or threonine.

70. The modified CFI polypeptide of aspect 68, wherein the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with tryptophan.

71. The modified CFI polypeptide of aspect 68, wherein the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with glutamine.

72. The modified CFI polypeptide of aspect 68, wherein the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with arginine.

73. The modified CFI polypeptide of aspect 68, wherein the mutation at a position corresponding to position 482 of SEQ ID NO: 1 is a substitution with threonine.

74. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 383 of SEQ ID NO: 1.

75. The modified CFI polypeptide of aspect 74, wherein:

76. The modified CFI polypeptide of aspect 74, wherein:

(ii) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, isoleucine, or tyrosine. 77. The modified CFI polypeptide of aspect 76, wherein:

78. The modified CFI polypeptide of aspect 76, wherein:

79. The modified CFI polypeptide of aspect 76, wherein:

80. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 401 of SEQ ID NO: 1.

81. The modified CFI polypeptide of aspect 80, wherein:

(ii) the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally H401Y. 82. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 409 of SEQ ID NO: 1.

83. The modified CFI polypeptide of aspect 82, wherein:

84. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 479 of SEQ ID NO: 1.

85. The modified CFI polypeptide of aspect 84, wherein:

86. The modified CFI polypeptide of aspect 74, wherein:

87. The modified CFI polypeptide of aspect 80, wherein: (i) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y; and

88. The modified CFI polypeptide of aspect 82, wherein:

89. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 401 of SEQ ID NO: 1.

90. The modified CFI polypeptide of aspect 89, wherein:

91. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 409 of SEQ ID NO: 1.

92. The modified CFI polypeptide of aspect 91, wherein: (i) the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally R383F; and

93. The modified CFI polypeptide of any one of the preceding aspects, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 383 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 439 of SEQ ID NO: 1.

94. The modified CFI polypeptide of aspect 93, wherein:

95. The modified CFI polypeptide according to any one of the preceding aspects, wherein the one or more mutations does not comprise a methionine to valine mutation at a position corresponding to position 138 of SEQ ID NO: 1.

96. The modified CFI polypeptide according to any one of the preceding aspects, wherein the one or more mutations does not comprise a mutation at a position corresponding to position 380 of SEQ ID NO: 1.

97. The modified CFI polypeptide according to any one of the preceding aspects, wherein the one or more mutations does not comprise a mutation at a position corresponding to position 429 of SEQ ID NO: 1.

98. The modified CFI polypeptide according to any one of the preceding aspects, wherein the one or more mutations does not comprise a mutation at a position corresponding to position 525 of SEQ ID NO: 1, optionally wherein the one or more mutations does not comprise a serine to alanine mutation at a position corresponding to position 525 of SEQ ID NO: 1.

99. The modified CFI polypeptide of any one of the preceding aspects, wherein the modified CFI polypeptide has higher CFI activity relative to a reference CFI polypeptide.

100. The modified CFI polypeptide of aspect 99, wherein the reference CFI polypeptide is a wild-type CFI polypeptide, optionally wherein the reference CFI polypeptide is the polypeptide of SEQ ID NO: 1.

101. The modified CFI polypeptide of aspect 99, wherein the reference CFI polypeptide is a wild-type CFI polypeptide, optionally wherein the reference CFI polypeptide is the polypeptide of SEQ ID NO: 2.

102. The modified CFI polypeptide of any one of aspects 99 to 101, wherein the CFI activity is at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, between 1.2 and 3 fold, between 1.2 and 5 fold, between 2 fold and 6 fold, between 3 fold and 8 fold, between 1.5 fold and 8 fold, between 2 fold and 8 fold, between 5 fold and 8 fold, between 6 fold and 8 fold, between 7 fold and 8 fold, between 2 fold and 15 fold, between 3 fold and 20 fold, between 5 fold and 10 fold, between 5 fold and 15 fold, or between 5 fold and 20 fold higher than the CFI activity of the reference CFI polypeptide.

103. The modified CFI polypeptide of any one of aspects 1 or 3 to 102, wherein the CFI activity is iC3b formation activity.

104. The modified CFI polypeptide of any one of aspects 1 or 3 to 103, wherein the CFI activity is determined using a homogeneous time resolved fluorescence (HTRF) assay.

105. The modified CFI polypeptide of any one of aspects 1 or 3 to 104, wherein the CFI activity is measured after 1 hour of incubation at pH 7 with C3b and CFH, followed by 1 hour of incubation with an anti-iC3b IgG and an anti-C3b IgG. 106. The modified CFI polypeptide of any one of aspects 1 or 3 to 105, wherein the CFI activity is measured after 30 minutes of incubation at pH 7 with C3b and CFH, followed by 30 minutes of incubation with an anti-iC3b IgG and an anti-C3b IgG.

107. The modified CFI polypeptide of aspect 105 or 106, wherein the anti-iC3b IgG is an anti-iC3b (Tb) IgG and the anti-C3b IgG is an anti-C3b (FITC) IgG.

108. The modified CFI polypeptide of any one of aspects 1, 3 to 104, 106 or 107, wherein the at least one mutation comprises:

(i) a mutation at a position which corresponds to position 53 of SEQ ID NO: 1 and is a substitution with leucine, optionally R53L; and

109. The modified CFI polypeptide of any one of the preceding aspects, wherein:

(i) the modified CFI polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 1 or SEQ ID NO: 2 of at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, between 400 and 565 amino acids, between 500 and 565 amino acids, between 520 and 565 amino acids, between 400 and 583 amino acids, between 500 and 583 amino acids, or between 520 and 583 amino acids; or

(ii) the modified CFI polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2; or (iii) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to a fragment of between 500 and 583 amino acids of SEQ ID NO: 1; or

(iv) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to a fragment of between 500 and 565 amino acids of SEQ ID NO: 2; or

(v) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1; or

(vi) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 2; or

(vii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises the one or more mutations defined in any one of the preceding aspects; or

(viii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1; or

(ix) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: l; or

(x) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 401 of SEQ ID NO: 1; or

(xi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 409 of SEQ ID NO: l; or (xii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 439 of SEQ ID NO: l; or

(xiii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 441 of SEQ ID NO: 1; or

(xiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 479 of SEQ ID NO: 1; or

(xv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 383 of SEQ ID NO: l; or

(xvi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 401 of SEQ ID NO: 1; or

(xvii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 409 of SEQ ID NO: l; or

(xviii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 53 of SEQ ID NO: 1 and a mutation at a position corresponding to position 479 of SEQ ID NO: 1; or

(xix) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 401 of SEQ ID NO: 1; or

(xx) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 439 of SEQ ID NO: 1; or

(xxii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with isoleucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxiii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1; or (xxiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with isoleucine at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxvi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxvii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1; or (xxviii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with aspartic acid at a position corresponding to position 479 of SEQ ID NO: 1; or

(xxix) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1; or (xxx) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1; or (xxxi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxxii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxxiii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 439 of SEQ ID NO: 1 ; or

(xxxiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1. 110. The modified CFI polypeptide of any one of the preceding aspects, wherein the modified CFI polypeptide comprises a signal peptide.

111. The modified CFI polypeptide of aspect 110, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a wild type CFI signal peptide.

112. The modified CFI polypeptide of aspect 110, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide.

113. The modified CFI polypeptide of aspect 110 or 112, wherein the signal peptide is a heterologous signal peptide.

114. The modified CFI polypeptide of any one of aspects 110, 112 or 113, wherein the signal peptide is from another polypeptide.

115. The modified CFI polypeptide of any one of aspects 110, 112 or 113, wherein the signal peptide is a modified CFI signal peptide.

116. The modified CFI polypeptide of any one of aspects 110, 112 or 113, wherein the signal peptide is a synthetic signal peptide.

117. A polynucleotide comprising a CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes the modified CFI polypeptide of any one of the preceding aspects.

118. The polynucleotide of aspect 117, wherein the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 16, 17, 18 or 19.

119. The polynucleotide of aspect 117 or 118, wherein the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a nucleotide sequence of any one of SEQ ID NOs: 16, 17, 18 or 19.

120. The polynucleotide of any one of aspects 117 to 119, wherein the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 18 or 19, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

121. The polynucleotide of any one of aspects 117 to 120, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1500 nucleotides of SEQ ID NO: 18 or 19.

122. The polynucleotide of any one of aspects 117 to 121, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 18 or 19.

123. The polynucleotide of aspect 117, wherein CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52. 124. The polynucleotide of aspect 117 or 123, wherein the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a nucleotide sequence of any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52.

125. The polynucleotide of any one of aspects 117, 123 or 124, wherein the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

126. The polynucleotide of any one of aspects 117 and 123 to 125, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1500 nucleotides of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

127. The polynucleotide of any one of aspects 117 and 123 to 126, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

128. The polynucleotide of any one of aspects 117 and 123 to 125, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1500 nucleotides of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, or 52. 129. The polynucleotide of any one of aspects 117, 123 to 125 and 128, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, or 52.

130. The polynucleotide of aspect 117, wherein the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 4 or 3.

131. The polynucleotide of aspect 130, wherein the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a nucleotide sequence of any one of SEQ ID NOs: 4 or 3.

132. The polynucleotide of any one of aspects 117, 130 and 131, wherein the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 4 or 3, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

133. The polynucleotide of any one of aspects 117 and 130 to 132, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at least 1500 nucleotides of SEQ ID NO: 4 or 3.

134. The polynucleotide of any one of aspects 117 and 130 to 133, wherein the CFI nucleotide sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 4 or 3. 135. The polynucleotide of any one of aspects 117 to 129, wherein at least a portion of the CFI nucleotide sequence is codon-optimised.

136. The polynucleotide of any one of aspects 117 to 129 and 135, wherein the CFI nucleotide sequence is codon-optimised.

137. The polynucleotide of aspect 135 or 136, wherein the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised is codon-optimised for expression in human liver cells.

138. The polynucleotide of aspect 135 or 136, wherein the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised is codon-optimised for expression in human retinal cells or human kidney cells.

139. The polynucleotide of any one of aspects 135 to 138, wherein the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised comprises a reduced number of CpGs compared to a corresponding portion of a reference CFI nucleotide sequence.

140. The polynucleotide of aspect 139, wherein the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised comprises 40 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer CpGs.

141. The polynucleotide of aspect 139 or 140, wherein the CFI nucleotide sequence comprises 5 or fewer CpGs.

142. The polynucleotide of any one of aspects 139 to 141, wherein the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised is CpG- free. 143. The polynucleotide of any one of aspects 139 to 142, wherein the CFI nucleotide sequence is CpG-free.

144. The polynucleotide of any one of aspects 139 to 143, wherein the reference CFI nucleotide sequence is a wild-type CFI nucleotide sequence.

145. The polynucleotide of aspect 144, wherein the wild-type CFI nucleotide sequence is the nucleotide sequence of SEQ ID NO: 3 or 4.

146. The polynucleotide of any one of aspects 117 to 145, wherein the CFI nucleotide sequence encodes a signal peptide.

147. The polynucleotide of aspect 146, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a wild type CFI signal peptide.

148 The polynucleotide of aspect 146, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide.

149. The polynucleotide of aspect 146 or 148, wherein the signal peptide is a heterologous signal peptide.

150. The polynucleotide of any one of aspects 146, 148, or 149, wherein the signal peptide is from another polypeptide.

151. The polynucleotide of any one of aspects 146, 148, or 149, wherein the signal peptide is a modified CFI signal peptide. 152. The polynucleotide of any one of aspects 146, 148, or 149, wherein the signal peptide is a synthetic signal peptide.

153. The polynucleotide of any one of aspects 117 to 152, wherein the polynucleotide further comprises a transcription regulatory element.

154. The polynucleotide of aspect 153, wherein the transcription regulatory element comprises a liver-specific promoter.

155. The polynucleotide of aspect 153 or 154, wherein the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11, 20, 21, or 22.

156. The polynucleotide of any one of aspects 153 to 155, wherein the transcription regulatory element has a sequence of SEQ ID NO: 11.

157. The polynucleotide of any one of aspects 153 to 155, wherein the transcription regulatory element has a sequence of SEQ ID NO: 20.

158. The polynucleotide of any one of aspects 153 to 155, wherein the transcription regulatory element has a sequence of SEQ ID NO: 21.

159. The polynucleotide of any one of aspects 153 to 155, wherein the transcription regulatory element has a sequence of SEQ ID NO: 22.

160. The polynucleotide of aspect 153, wherein the transcription regulatory element comprises an ocular-specific promoter and/or enhancer.

161. The polynucleotide of aspect 153, wherein the transcription regulatory element comprises a retinal cell-specific promoter and/or enhancer. 162. The polynucleotide of aspect 153, wherein the transcription regulatory element comprises a kidney-specific promoter and/or enhancer.

163. The polynucleotide of aspect 153, wherein the transcription regulatory element comprises a podocyte cell-specific promoter and/or enhancer.

164. The polynucleotide of aspect 153, wherein the transcription regulatory element is a non-tissue-specific transcription regulatory element.

165. The polynucleotide of any one of aspects 117 to 164, wherein the polynucleotide comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE).

166. The polynucleotide of any one of aspects 117 to 165, wherein the polynucleotide comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to any one of SEQ ID NOs: 23 to 25.

167. The polynucleotide of any one of aspects 117 to 166, wherein the polynucleotide comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) at least 98% identical to any one of SEQ ID NOs: 23 to 25.

168. The polynucleotide of any one of aspects 117 to 167, wherein the polynucleotide comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) of any one of SEQ ID NOs: 23 to 25.

169. The polynucleotide of any one aspects 117 to 168, wherein the polynucleotide further comprises one or two ITR(s).

170. The polynucleotide of aspect 169, wherein the or each ITR is a wild-type ITR. 171. The polynucleotide of aspect 169 or 170, wherein the or each ITR is an AAV2 ITR.

172. The polynucleotide of any one of aspects 169 to 171, wherein the nucleotide sequence of the or each ITR comprises a nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

173. The polynucleotide of any one of aspects 117 to 172, wherein the polynucleotide further comprises a polyA nucleotide sequence.

174. The polynucleotide of aspect 173, wherein the polyA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 14.

175. The polynucleotide of any one aspects 117 to 174, wherein the polynucleotide further comprises an intron.

176. The polynucleotide of aspect 117, wherein the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

177. The polynucleotide of aspect 117 or 176, wherein the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3 or 5 from W02020/086735 or SEQ ID NO: 8 from WO2017/072515, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide has one or more, two or more, three of more, four or more, or five or more amino acid substitutions at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

178. The polynucleotide of aspect 117, wherein the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 44 to 52, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide comprises the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

179. The polynucleotide of aspect 117 or 178, wherein the CFI nucleotide sequence is the nucleotide sequence of any one of SEQ ID NOs: 44 to 52, except that it comprises nucleotide substitutions such that the encoded CFI polypeptide has one or more, two or more, three of more, four or more, or five or more amino acid substitutions at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

180. The polynucleotide of any one of aspects 176 to 179, wherein the encoded CFI polypeptide has (i) a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1, optionally R53L; and (ii) a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1, optionally R383Y.

181. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of aspects 117 to 180.

182. The viral particle of aspect 181, which is an AAV, adenoviral, or lentiviral viral particle.

183. The viral particle of aspect 182, which is an AAV viral particle. 184. The viral particle of any one of aspects 181 to 183, wherein the viral particle comprises a capsid.

185. The viral particle of aspect 184, wherein the viral particle comprises a liver-tropic capsid.

186. The viral particle of aspect 185, wherein the liver-tropic capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and 734, between 700 and 736, around 734, or around 736 amino acids of any one of SEQ ID NOs: 7-10.

187. The viral particle of aspects 185 or 186, wherein the liver-tropic capsid comprises a sequence at least 99%, or 100% identical to any one of SEQ ID NOs: 7-10.

188. The viral particle of aspect 184, wherein the capsid comprises a sequence at least 99%, or 100% identical to SEQ ID NO: 26 or 27.

189. The viral particle of aspect 184, wherein the capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and 734, between 700 and 736, around 734, or around 736 amino acids of any one of SEQ ID NOs: 7-10.

190. The viral particle of aspect 184 or 189, wherein the capsid comprises a sequence at least 99%, or 100% identical to any one of SEQ ID NOs: 7-10.

191. The viral particle of aspect 184, wherein the viral particle comprises a kidneytropic capsid. 192. The viral particle of any one of aspects 181 to 191, wherein the recombinant genome is single-stranded.

193. The viral particle of any one of aspects 181 to 192, wherein the viral particle further comprises: a) AAV2 ITRs; b) a polyA sequence; and/or c) an intron.

194. A composition comprising the modified CFI polypeptide, polynucleotide or viral particle of any one of the preceding aspects, and a pharmaceutically acceptable excipient.

195. The modified CFI polypeptide according to any one of aspects 1 to 116, the polynucleotide according to any one of aspects 117 to 180, the viral particle according to any one of aspects 181 to 193, or the composition according to aspect 194 for use in a method of treatment.

196. A method of treatment comprising administering an effective amount of the modified CFI polypeptide according to any one of aspects 1 to 116, the polynucleotide according to any one of aspects 117 to 180, the viral particle according to any one of aspects 181 to 193, or the composition according to aspect 194.

197. Use of the modified CFI polypeptide according to any one of aspects 1 to 116, the polynucleotide according to any one of aspects 117 to 180, the viral particle according to any one of aspects 181 to 193, or the composition according to aspect 194 in the manufacture of a medicament for use in a method of treatment.

198. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, method, or use of any one of aspects 195 to 197, wherein the method of treatment comprises administering an effective amount of the polypeptide, polynucleotide, viral particle or composition to a patient.

199. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, method, or use of any one of aspects 195 to 198, wherein the method of treatment is a method of gene therapy.

200. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, method, or use of any one of aspects 195 to 199, wherein the method of treatment is a method of treatment by increasing expression of the CFI polypeptide.

201. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, or use of any one of aspects 195 to 200, wherein the method of treatment is a method of treating a complement-mediated disorder.

202. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of aspect 201, wherein the disorder is a C3 -mediated disorder.

203. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of aspect 201 or 202, wherein the disorder is a kidney disorder.

204. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 203, wherein the disorder is associated with over-activity of the complement C3b feedback cycle.

205. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 204, wherein the disorder is selected from C3 glomerulopathy, IgA nephropathy, lupus nephritis, systemic lupus erythematosus, membranous nephropathy, membranoproliferative glomerulonephritis, paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome, autoimmune haemolytic anaemia, ANCA-associated vasculitis, Gaucher disease, peritonitis, age-related macular degeneration, diabetic retinopathy, dense deposit disease, age-related inflammatory or autoinflammatory diseases, autoimmune arthritis such as rheumatoid arthritis, atherosclerosis, chronic cardiovascular disease, Alzheimer’s disease, systemic vasculitis, Guillain-Barre syndrome, and Henoch-Schonlein purpura.

206. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205, wherein the disorder is selected from C3 glomerulopathy, C3 glomerulonephritis and dense deposit disease.

207. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205, wherein the disorder is atypical haemolytic uremic syndrome, optionally atypical haemolytic uremic syndrome with monoallelic CFH mutation.

208. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205, wherein the disorder is a kidney glomerular or tubular disorder.

209. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205 or 208, wherein the disorder is selected from C3 glomerulopathy, IgA nephropathy, lupus nephritis and membranous nephropathy.

210. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205, 208 or 209, wherein the disorder is lupus nephritis.

211. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 205, wherein the disorder is systemic lupus erythematosus. 212. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 211, wherein the polypeptide, polynucleotide, viral particle, or composition is according to any one of aspects 1 to 194.

213. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 202, wherein the method of treatment is a method of treating an ocular disorder.

214. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201, 202 and 204, wherein the disorder is an ocular disorder.

215. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 214, wherein the method of treatment reduces the rate of, or prevents, the progression of a degenerative-type disorder.

216. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 213 to 215, wherein the ocular disorder is AMD, diabetic retinopathy, glaucoma, Stargardt’s disease, central serous chorioretinopathy, retinitis pigmentosa, diabetic macular edema (DME), keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, uveitis, vitreoretinopathy, or is non-oncological.

217. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 213 to 216, wherein the ocular disorder is AMD.

218. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of aspect 216 or 217, wherein the AMD is dry AMD or geographic atrophy (GA). 219. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 202, 204 and 213 to 218, wherein the method of treatment is a method of treating AMD, dry AMD or GA.

220. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 202, 204 and 213 to 219, wherein the method of treatment results in the treatment of GA.

221. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 220, wherein the method of treatment results in (i) a prevention of an increase in the area of GA, or (ii) a reduction in the area of GA.

222. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 221, wherein the method of treatment results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in the amount of GA at least 12 months from the administration of the CFI polypeptide, polynucleotide, viral particle and/or composition to an eye, optionally wherein the amount of GA is the area of GA.

223. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 222, wherein the method of treatment results in a prevention of, or reduction in the rate of, progression of GA.

224. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 223, wherein the method of treatment results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in the rate of increase in GA area at least 12 months from the administration of the CFI polypeptide, polynucleotide, viral particle and/or composition to an eye, relative to an untreated eye over the same period.

225. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 224, wherein the method of treatment results in a prevention of, or reduction in the rate of, formation of drusen.

226. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 225, wherein the method of treatment results in a reduction in existing drusen, optionally a reduction in the size and/or number of existing drusen.

227. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 226, wherein the method of treatment results in a prevention of, or reduction in the rate of, complement deposition.

228. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 217 to 227, wherein the method of treatment results in a reduction in existing complement deposition.

229. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 213 to 228, wherein the polypeptide, polynucleotide, viral particle, or composition is administered by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection.

230. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 213 to 229, wherein the polypeptide, polynucleotide, viral particle, or composition is administered intraocularly. 231. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 212, wherein the disorder is not an ocular disorder.

232. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of aspect 231, wherein the disorder is a systemic acute disorder.

233. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of aspect 231, wherein the disorder is a systemic chronic disorder.

234. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 212, or 231 to 233, wherein the polypeptide, polynucleotide, viral particle, or composition is not administered by subretinal injection, direct retinal injection, suprachoroidal injection, and/or intravitreal injection.

235. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 201 to 212, or 231 to 234, wherein the polypeptide, polynucleotide, viral particle, or composition is not administered intraocularly.

236. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 228, or 231 to 235, wherein the polynucleotide, viral particle, or composition is administered: intravenously; systemically; to the liver via peripheral vein infusion; to the liver via hepatic vessels such as hepatic vein infusion or hepatic artery infusion; or via intraparenchymal administration direct to the liver.

237. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 228, or 231 to 235, wherein the polypeptide, polynucleotide, viral particle, or composition is administered by injection into the renal artery.

238. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 228, or 231 to 235, wherein the polypeptide, polynucleotide, viral particle, or composition is administered by retrograde administration, optionally wherein the administration is via the ureters using a urinary catheter.

239. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 228, or 231 to 235, wherein the polypeptide, polynucleotide, viral particle, or composition is administered via direct kidney parenchymal injection or sub-capsular injection.

240. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 239, wherein administration of the polypeptide, polynucleotide, viral particle, or composition increases the level of C3b- inactivating and/or iC3b-degradation activity in the patient.

241. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of aspect 240, wherein administration of the polypeptide, polynucleotide, viral particle or composition increases the level of C3b-inactivating and/or iC3b-degradation activity above a normal level.

242. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of aspect 240 or 241, wherein administration of the polypeptide, polynucleotide, viral particle or composition increases the level of C3b-inactivating and/or iC3b-degradation activity to a level 1.2-fold, 1.5-fold, 1.8-fold, or 2-fold higher than the normal level. 243. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of aspect 241 or 242, wherein a normal level is equivalent to that provided by 30-40pg/ml of CFI polypeptide in the serum.

244. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 195 to 243, wherein the method of treatment is a method for increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient.

245. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 195 to 244, wherein the method of treatment is a method for increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient, thereby treating the complement-mediated disorder.

246. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 195 to 245, wherein the method of treatment is a method for treating a disease or disorder by increasing the level of C3b-inactivating and/or iC3b-degradation activity in a patient.

247. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 244 to 246, wherein increasing the level of C3b- inactivating and/or iC3b-degradation activity in a patient is increasing the level of C3b- inactivating and/or iC3b-degradation activity above a normal level.

248. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 244 to 247, wherein increasing the level of C3b- inactivating and/or iC3b-degradation activity in a patient is increasing the level of C3b- inactivating and/or iC3b-degradation activity to a level 1.2-fold, 1.5-fold, 1.8-fold, or 2- fold higher than the normal level. 249. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 240 to 248, wherein the increase in the level of C3b-inactivating and/or iC3b-degradation activity is an increase in the level of C3b- inactivating and/or iC3b-degradation activity in the tissue or organ to which the CFI polypeptide, polynucleotide, viral particle, or composition is administered.

250. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 198 to 249, wherein the patient has a lower than normal level of CFI protein.

251. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 198 to 250, wherein the patient has a lower than normal level of CFI expression and/or a lower than normal level of CFI activity.

252. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 198 to 249, wherein the patient has a normal level of CFI protein.

253. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 198 to 249 and 252, wherein the patient has a normal level of CFI expression and/or a normal level of CFI activity.

254. The modified CFI polypeptide, polynucleotide, viral particle or composition for use, use, or method of any one of aspects 247 to 253, wherein a normal level is equivalent to that provided by 30-40pg/ml of CFI polypeptide in the serum.

255. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, use, or method of any one of aspects 195 to 254, wherein the polypeptide, polynucleotide, viral particle, or composition is according to any one of aspects 1 to 194.

Claims

3. The modified CFI polypeptide of claim 1 or 2, wherein the one or more mutations comprises:

4. The modified CFI polypeptide of claim 3, wherein at least one of the positions within the modified CFI polypeptide:

(i) which can form an interface with C3b is in the SP domain;

(ii) which can form an interface with C3b is in the FIM domain; and/or

(iii) which can form an interface with Factor H is in the SP domain.

5. The modified CFI polypeptide of claim 3 or 4, wherein:

(i) the interface with C3b is the interface with the C3b alpha chain;

(ii) the interface with C3b is the interface with the CUB domain in the C3b alpha chain;

(iii) the interface with C3b is the interface with the substrate loop within the CUB domain in the C3b alpha chain; (iv) the interface with C3b is the interface with the CTC domain in the C3b alpha chain;

(v) the interface with Factor H is the interface with the CCP2 domain in Factor H; and/or

(vi) the interface with Factor H is the interface with the CCP3 domain in Factor H.

6. The modified CFI polypeptide of any one of the preceding claims, wherein the one or more mutations comprises a mutation at one or more, two or more, three or more, four or more, or five or more positions corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1.

7. The modified CFI polypeptide of any one of the preceding claims, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1, optionally wherein the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine, or glutamic acid;

(ii) a mutation at a position corresponding to position 383 of SEQ ID NO: 1, optionally wherein the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, leucine, tyrosine, glutamic acid, or phenylalanine;

(iii) a mutation at a position corresponding to position 401 of SEQ ID NO: 1, optionally wherein the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with glycine, tyrosine, phenylalanine, or tryptophan, further optionally tyrosine, phenylalanine, or tryptophan;

(iv) a mutation at a position corresponding to position 409 of SEQ ID NO: 1, optionally, wherein the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, tryptophan, or glutamic acid;

(v) a mutation at a position corresponding to position 439 of SEQ ID NO: 1, optionally wherein the mutation at a position corresponding to position 439 of SEQ ID NO: 1 is a substitution with leucine, glutamic acid or glutamine; and/or

(vi) a mutation at a position corresponding to position 479 of SEQ ID NO: 1, optionally wherein the mutation at a position corresponding to position 479 of SEQ ID NO: 1 is a substitution with aspartic acid, optionally E479D.

8. The modified CFI polypeptide of any one of the preceding claims, wherein the one or more mutations comprises:

(i) a mutation at a position corresponding to position 53 of SEQ ID NO: 1; and

(ii) a mutation at a position corresponding to position 383, 401, or 409 of SEQ ID NO: 1, optionally, wherein:

(a) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L, and the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R383Y;

(b) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R53I, and the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with glutamic acid, optionally R383E;

(c) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L, and the mutation at a position corresponding to position 383 of SEQ ID NO: 1 is a substitution with isoleucine, optionally R383I;

154 (d) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with leucine, optionally R53L, and the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R401Y;

(e) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y, and the mutation at a position corresponding to position 401 of SEQ ID NO: 1 is a substitution with tyrosine, optionally H401Y; or

(1) the mutation at a position corresponding to position 53 of SEQ ID NO: 1 is a substitution with tyrosine, optionally R53Y, and the mutation at a position corresponding to position 409 of SEQ ID NO: 1 is a substitution with phenylalanine, optionally I409F.

9. The modified CFI polypeptide of any one of the preceding claims, wherein the modified CFI polypeptide has higher CFI activity relative to a reference CFI polypeptide.

10. The modified CFI polypeptide of claim 9, wherein the reference CFI polypeptide is a wild-type CFI polypeptide, further optionally wherein the reference CFI polypeptide is the polypeptide of SEQ ID NO: 1 or 2.

11. The modified CFI polypeptide of claim 9 or 10, wherein the CFI activity is at least 1.2 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, between 1.2 and 3 fold, between 1.2 and 5 fold, between 2 fold and 6 fold, between 3 fold and 8 fold, between 1.5 fold and 8 fold, between 2 fold and 8 fold, between 5 fold and 8 fold, between 6 fold and 8 fold, between 7 fold and 8 fold, between 2 fold and 15 fold, between 3 fold and 20 fold, between 5 fold and 10 fold, between 5 fold and 15 fold, or between 5 fold and 20 fold higher than the CFI activity of the reference CFI polypeptide.

12. The modified CFI polypeptide of any one of claims 1 or 3 to 11, wherein the CFI activity is iC3b formation activity.

13. The modified CFI polypeptide of any one of claims 1 or 3 to 12, wherein the CFI activity is determined using a homogeneous time resolved fluorescence (HTRF) assay.

155

14. The modified CFI polypeptide of any one of claims 1 or 3 to 13, wherein the CFI activity is measured after 1 hour of incubation at pH 7 with C3b and CFH, followed by 1 hour of incubation with an anti-iC3b IgG and an anti-C3b IgG.

15. The modified CFI polypeptide of any one of claims 1 or 3 to 13, wherein the CFI activity is measured after 30 minutes of incubation at pH 7 with C3b and CFH, followed by 30 minutes of incubation with an anti-iC3b IgG and an anti-C3b IgG.

16. The modified CFI polypeptide of claim 14 or 15, wherein the anti-iC3b IgG is an anti-iC3b (Tb) IgG and the anti-C3b IgG is an anti-C3b (FITC) IgG.

17. The modified CFI polypeptide of any one of the preceding claims, wherein:

(ii) the modified CFI polypeptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2; or

(iii) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to a fragment of between 500 and 583 amino acids of SEQ ID NO: 1; or

(v) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1; or (vi) the modified CFI polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO: 2; or

(vii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises the one or more mutations defined in any one of the preceding claims; or

(xi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 409 of SEQ ID NO: l; or

(xii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 439 of SEQ ID NO: l; or

(xiii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 441 of SEQ ID NO: 1; or (xiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 479 of SEQ ID NO: 1; or

158 (xx) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a mutation at a position corresponding to position 383 of SEQ ID NO: 1 and a mutation at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxiii) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with glutamic acid at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with isoleucine at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position

159 corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1; or (xxvi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with leucine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxix) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 383 of SEQ ID NO: 1; or

(xxx) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1; or

160 (xxxi) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with tyrosine at a position corresponding to position 53 of SEQ ID NO: 1 and a substitution with phenylalanine at a position corresponding to position 409 of SEQ ID NO: 1; or

(xxxiv) the modified CFI polypeptide comprises an amino acid sequence that is identical to SEQ ID NO: 1 or SEQ ID NO: 2, except that the modified CFI polypeptide comprises a substitution with phenylalanine at a position corresponding to position 383 of SEQ ID NO: 1 and a substitution with tyrosine at a position corresponding to position 401 of SEQ ID NO: 1.

18. The modified CFI polypeptide of any one of the preceding claims, wherein:

(i) the modified CFI polypeptide comprises a signal peptide; or

(ii) the modified CFI polypeptide comprises a signal peptide, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a wild type CFI signal peptide; or

161 (iii) the modified CFI polypeptide comprises a signal peptide, wherein the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide; or

(iv) the modified CFI polypeptide comprises a signal peptide, wherein the signal peptide is a heterologous signal peptide; or

(v) the modified CFI polypeptide comprises a signal peptide, wherein the signal peptide is from another polypeptide; or

(vi) the modified CFI polypeptide comprises a signal peptide, wherein the signal peptide is a modified CFI signal peptide; or

(vii) the modified CFI polypeptide comprises a signal peptide, wherein the signal peptide is a synthetic signal peptide.

19. A polynucleotide comprising a CFI nucleotide sequence, wherein the CFI nucleotide sequence encodes the modified CFI polypeptide of any one of the preceding claims.

20. The polynucleotide of claim 19, wherein:

(i) the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 16, 17, 18 or 19;

(ii) the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 28, 29, 30, 31, 32,

162 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52.

(iii) the CFI nucleotide sequence comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical to a fragment of at least 1200, at least 1400, at least 1500, 1698 or fewer, 1752 or fewer, between 1200 and 1698, between 1200 and 1752, between 1500 and 1698, between 1500 and 1752, around 1698, or around 1752 nucleotides of any one of SEQ ID NOs: 4 or 3;

(iv) the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 18 or 19, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1;

(v) the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1;

(vi) the CFI nucleotide sequence comprises a sequence that is 100% identical to SEQ ID NO: 4 or 3, except for the codon encoding the one or more mutations providing a higher CFI activity or the one or more mutations comprising a mutation at a position corresponding to a position selected from the group consisting of 53, 60, 383, 384, 400, 401, 407, 408, 409, 439, 440, 441, 479, and 482 of SEQ ID NO: 1;

(vii) the CFI nucleotide sequence or a portion of the CFI nucleotide sequence that is codon-optimised is codon-optimised for expression in human liver cells, human retinal cells or human kidney cells;

163 (viii) the CFI nucleotide sequence or the portion of the CFI nucleotide sequence that is codon-optimised comprises a reduced number of CpGs compared to a corresponding portion of a reference CFI nucleotide sequence, optionally wherein the reference CFI nucleotide sequence is a wild-type CFI nucleotide sequence;

(ix) the CFI nucleotide sequence comprises 5 or fewer CpGs;

(x) the CFI nucleotide sequence encodes a signal peptide;

(xi) the CFI nucleotide sequence encodes a signal peptide, and the amino acid sequence of the signal peptide is an amino acid sequence of a signal peptide which is not a wild type CFI signal peptide;

(xii) the CFI nucleotide sequence encodes a signal peptide, and the signal peptide is a heterologous signal peptide; and/or

(xiii) the polynucleotide further comprises a transcription regulatory element, optionally wherein the transcription regulatory element comprises (a) a liver-specific promoter, (b) an ocular-specific promoter and/or enhancer, (c) a retinal cell-specific promoter and/or enhancer, (d) a kidney-specific promoter and/or enhancer, (e) a podocyte cell-specific promoter and/or enhancer, or (f) a non-tissue-specific transcription regulatory element.

21. A viral particle comprising a recombinant genome comprising the polynucleotide of claim 19 or 20.

22. The viral particle of claim 21 :

(i) which is an AAV, adenoviral, or lentiviral viral particle;

(ii) which is an AAV viral particle;

(iii) wherein the viral particle comprises a capsid;

(iv) wherein the viral particle comprises a capsid and the capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between

164 700 and 734, between 700 and 736, around 734, or around 736 amino acids of any one of SEQ ID NOs: 7-10;

(v) wherein the viral particle comprises a liver-tropic capsid;

(vi) wherein the viral particle comprises a liver-tropic capsid and the liver-tropic capsid comprises a sequence at least 98%, at least 99%, at least 99.5%, or 100% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 734, between 600 and 736, between 650 and 734, between 650 and 736, between 700 and 734, between 700 and 736, around 734, or around 736 amino acids of any one of SEQ ID NOs: 7-10;

(vii) wherein the viral particle comprises a capsid and the capsid comprises a sequence at least 99%, or 100% identical to SEQ ID NO: 26 or 27;

(viii) wherein the viral particle comprises a kidney-tropic capsid;

(ix) wherein the recombinant genome is single-stranded; and/or

(x) wherein the viral particle further comprises: a) AAV2 ITRs; b) a polyA sequence; and/or c) an intron.

23. A composition comprising the modified CFI polypeptide, polynucleotide or viral particle of any one of the preceding claims, and a pharmaceutically acceptable excipient.

24. The modified CFI polypeptide according to any one of claims 1 to 18, the polynucleotide according to claim 19 or 20, the viral particle according to claim 21 or 22, or the composition according to claim 23 for use in a method of treatment, optionally wherein the method of treatment comprises administering an effective amount of the polypeptide, polynucleotide, viral particle or composition to a patient.

25. A method of treatment comprising administering an effective amount of the modified CFI polypeptide according to any one of claims 1 to 18, the polynucleotide according to claim 19 or 20, the viral particle according to claim 21 or 22, or the

165 composition according to claim 23, optionally wherein the method of treatment comprises administering an effective amount of the polypeptide, polynucleotide, viral particle or composition to a patient.

26. Use of the modified CFI polypeptide according to any one of claims 1 to 18, the polynucleotide according to claim 19 or 20, the viral particle according to claim 21 or 22, or the composition according to claim 23 in the manufacture of a medicament for use in a method of treatment, optionally wherein the method of treatment comprises administering an effective amount of the polypeptide, polynucleotide, viral particle or composition to a patient.

27. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, method, or use according to any one of claims 24 to 26, wherein the method of treatment:

(i) is a method of gene therapy;

(ii) is a method of treatment by increasing expression of the CFI polypeptide;

(iii) is a method of treating a complement-mediated disorder; and/or

(iv) is a method of treating an ocular disorder.

28. The modified CFI polypeptide, polynucleotide, viral particle, or composition for use, method, or use according to any one of claims 24 to 27, wherein the complement- mediated disorder is:

(i) a C3 -mediated disorder;

(ii) a kidney disorder;

(iii) associated with over-activity of the complement C3b feedback cycle;

(iv) selected from C3 glomerulopathy, IgA nephropathy, lupus nephritis, systemic lupus erythematosus, membranous nephropathy, membranoproliferative glomerulonephritis, paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome, autoimmune haemolytic anaemia, ANCA-associated vasculitis, Gaucher disease,

166 peritonitis, age-related macular degeneration including dry AMD and geographic atrophy, diabetic retinopathy, dense deposit disease, age-related inflammatory or autoinflammatory diseases, autoimmune arthritis such as rheumatoid arthritis, atherosclerosis, chronic cardiovascular disease, Alzheimer’s disease, systemic vasculitis, Guillain-Barre syndrome, and Henoch-Schonlein purpura; and/or

(v) a kidney glomerular or tubular disorder.