Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

Experimental confirmation of the C3 tickover hypothesis by studies with an Ab (S77) that inhibits tickover in whole serum.

The complement component 3 (C3) tickover hypothesis was put forward in the early 1970s to account for the spontaneous activation of the alternative complement pathway that occurs after the genetic absence or in vitro depletion of Factor I, the enzyme that is essential for the breakdown of C3b. The hypothesis was widely accepted, but experimental demonstration of the tickover was elusive. A phage Ab against C3b that inhibited the alternative complement pathway, but not the classical pathway, was described in 2009. Studies using this Ab in a variety of assays have now demonstrated that it acts primarily by inhibiting tickover, thereby confirming that tickover really exists.-Lachmann, P. J., Lay, E., Seilly, D. J. Experimental confirmation of the C3 tickover hypothesis by studies with an Ab (S77) that inhibits tickover in whole serum.

Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

Low-dose recombinant properdin provides substantial protection against Streptococcus pneumoniae and Neisseria meningitidis infection.

Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (Pn), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae. In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of Pn (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the Pn treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of Pn treated mice presented with sepsis and recoverable levels of live Meningococci. In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of Pn at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of Pn administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.

Conversion of the Liver into a Biofactory for DNaseI Using Adeno-Associated Virus Vector Gene Transfer Reduces Neutrophil Extracellular Traps in a Model of Systemic Lupus Erythematosus.

Adeno-associated virus (AAV) vectors are proving to be clinically transformative tools in the treatment of monogenic genetic disease. Rapid ongoing development of this technology promises to not only increase the number of monogenic disorders amenable to this approach but also to bring diseases with complex multigenic and nongenetic etiologies within therapeutic reach. In this study, we explore the broader paradigm of converting the liver into a biofactory for systemic output of therapeutic molecules using AAV-mediated delivery of the endonuclease DNaseI as an exemplar. DNaseI can clear neutrophil extracellular traps (NETs), which are nuclear-protein structures possessing antimicrobial action, also involved in the pathophysiology of clinically troubling immune-mediated diseases. However, a translational challenge is short half-life of the enzyme in vivo (<5 h). This study demonstrates that AAV-mediated liver-targeted gene transfer stably induces serum DNaseI activity to >190-fold above physiological levels. In lupus-prone mice (NZBWF1), the activity was maintained for longer than 6 months, the latest time point tested, and resulted in a clear functional effect with reduced renal presence of neutrophils, NETs, IgG, and complement C3. However, treatment in this complex disease model did not extend lifespan, improve serological endpoints, or preserve renal function, indicating there are elements of pathophysiology not accessible to DNaseI in the NZBWF1 model. We conclude that a translational solution to the challenge of short half-life of DNaseI is AAV-mediated gene delivery and that this may be efficacious in treating disease where NETs are a dominant pathological mechanism.

The grandmother effect.

Two classes of explanation have been advanced to explain women's long post-menopausal life span - that it is an epiphenomenon of increased longevity or an evolved adaptation. While there is no decisive evidence to choose between them, the latter appears more likely. Two mechanisms for the evolved adaptation have been proposed, the 'mother effect' and the 'grandmother effect'. It is argued that both these effects, insofar as they have been shown to occur, are likely to be culturally rather than genetically evolved.

Adeno-Associated Virus Vector Gene Delivery Elevates Factor I Levels and Downregulates the Complement Alternative Pathway In Vivo.

The complement system is a key component of innate immunity, but impaired regulation influences disease susceptibility, including age-related macular degeneration and some kidney diseases. While complete complement inhibition has been used successfully to treat acute kidney disease, key unresolved challenges include strategies to modulate rather than completely inhibit the system and to deliver therapy potentially over decades. Elevating concentrations of complement factor I (CFI) restricts complement activation in vitro and this approach was extended in the current study to modulate complement activation in vivo. Sustained increases in CFI levels were achieved using an adeno-associated virus (AAV) vector to target the liver, inducing a 4- to 5-fold increase in circulating CFI levels. This led to decreased activity of the alternative pathway as demonstrated by a reduction in the rate of inactive C3b (iC3b) deposition and more rapid formation of C3 degradation products. In addition, vector application in a mouse model of systemic lupus erythematosus (NZBWF1), where tissue injury is, in part, complement dependent, resulted in reduced complement C3 and IgG renal deposition. Collectively, these data demonstrate that sustained elevation of CFI reduces complement activation in vivo providing proof-of-principle support for the therapeutic application of AAV gene delivery to modulate complement activation.

The story of complement factor I.

Factor I was first discovered in 1966. Its importance became apparent with the description of the original Factor I deficient patient in Boston in 1967. This patient presented with a hyperactive alternative complement pathway resulting in secondary complement deficiency due to continuous complement consumption. On the basis of these findings, the mechanism of the alternative pathway was worked out. In 1975, the surprise finding was made that elevating levels of Factor I in plasma down-regulated the alternative pathway. Attempts to exploit this finding for clinical use had a long and frustrating history and it was not until 2019 that the first patient was treated with the gene therapy vector for age related macular degeneration by Professor Sir Robert MacLaren in Oxford. This review follows the long and contorted course from initial observations to clinical use of complement Factor I.

Looking back on the alternative complement pathway.

The alternative pathway of complement originated from the Properdin pathway originally described by the Pillemer laboratory in the 1950s. This work generated great controversy and it took several decades for a consensus on its components, its reaction sequence and its functions to emerge. This paper reviews this history and attempts to clarify some of the ambiguities that remain.

A novel and sensitive functional assay for complement Factor I based on the third proteolytic clip of C3b.

A sensitive assay for the functional activity of complement Factor I is described. This is based on its third proteolytic clip whereby Factor I cleaves cell-bound iC3b to cell-bound C3dg and soluble C3c, thereby abolishing conglutination of the cells. Factor H is required as a co-factor for Factor I activity. Because of the low affinity of iC3b for Factor H, the assay needs to be performed at low ionic strength. This assay is easier to perform than those based on the conversion of C3b to iC3b (the first two Factor I clips), there being no need for the unstable intermediate EAC142 or for purified C3.

Streptococcal DRS (distantly related to SIC) and SIC inhibit antimicrobial peptides, components of mucosal innate immunity: a comparison of their activities.

"Streptococcal inhibitor of complement" (SIC) and "distantly related to SIC" (DRS) are related virulence factors secreted by M1 and M12 strains of GAS, respectively. The human mucosal innate immune system, important components of which are beta-defensins, secretory leukocyte proteinase inhibitor (SLPI) and lysozyme, provides the first line of defence against microorganisms. We report the interaction between DRS and these proteins; further investigations into the interaction of SIC with the beta-defensins; and compare the sensitivity of M12 and M1 GAS to SLPI. We show that SLPI, which kills M1 GAS and is inhibited by SIC, cannot kill M12 GAS. DRS cannot inhibit SLPI killing of M1 GAS, although ELISA shows binding of DRS to SLPI. We suggest that the target for SLPI on M1 GAS resembles SIC, and soluble SIC inhibits by acting as a decoy for SLPI. M12 GAS may not have this target and cannot interact with SLPI. DRS inhibits the antibacterial action of hBD-2 and hBD-3. Binding of both SIC and DRS to hBD-2, and DRS to hBD-3, shows small positive enthalpy, suggesting that binding is largely hydrophobic. The data for SIC and hBD-3 indicate that this is not a homogeneous bimolecular interaction. We conclude that DRS shares several of the properties of SIC, and therefore can be considered an important virulence factor of M12 GAS and an aid to colonization of the host mucosae.

A more radical solution.

The current modifications to licensing procedures still leave a basically flawed system in place. A more radical solution is proposed that involves dispensing with Phase 3 trials and making medicines available at the end of Phase 2 to those who are fully informed of the potential risks and benefits and wish to take part in this novel procedure. The advantages include a shorter development time, lower development costs and allowing smaller companies to take medicines to the clinic. The principal obstacle is that medicines are subject to strict liability rather than the tort of negligence - and this will have to be amended in due course.

Stem cell therapy: medical advance or moral challenge?

My professional lifetime has seen progress in the biomedical sciences that beggars belief. This has lead to astonishing advances in the ability to prevent and treat disease and, in the developed world at least, people live longer and healthier lives than ever before. Paradoxically, this has gone hand in hand with the growth of a vocal and influential anti-science lobby that not only rejects much modern science but is also deeply suspicious of new medical interventions. The prospect of cell therapy in the near or middle future is their current target especially where the use of embryonic stem cells or of cell nuclear transfer techniques is concerned. The prospect of cell therapy is welcomed with enthusiasm by patients with genetic and degenerative diseases who hope to benefit from them. On the other hand the whole idea is regarded as repugnant by the anti-science lobby. While some of this opposition is essentially luddite in nature, there are some more persuasive arguments raised particularly to any research than uses embryonic or foetal materials. These arguments will be examined critically. The moral problems of denying the sick the hope of effective treatments have to be weighed against those seen in the development of such treatments. (This article is closely based on an already published paper. P. Lachmann, Stem cell research: why is it regarded as a threat? An investigation of the economic and ethical arguments made against research with human embryonic stem cells.

The use of antibodies in the prophylaxis and treatment of infections.

The use of antibodies to provide passive immunity to infections has a long history. Although the coming of antibiotics greatly reduced its use for bacterial infections, it is still widely used for a variety of purposes which are reviewed here. The use of animal antisera gave way to the use of human convalescent serum as a source of antibodies and more recently human and monoclonal antibodies have become widely used, not just providing passive immunity but as therapeutic agents. The current uses of antibody therapy are discussed as are the problems of antibody-mediated immunopathology and how this can be avoided. More recent developments include the making of monoclonal antibodies that react with cross-reacting determinants on flu viruses. Such antibodies are not usually made following infection and they provide a very promising approach to providing passive immunity that will be effective against a variety of different strains of the flu virus. It is also pointed out that passive immunotherapy can act as a surrogate vaccine providing that the subject gets infected while protected by the passive antibodies. Finally, there is a section on the possible use of oral antibodies given as food to prevent diseases such as infantile gastroenteritis.