Circumventing B Cell Responses to Allow for Redosing of Adeno-Associated Virus Vectors

Abstract

Adeno-associated virus (AAV)-mediated gene therapy has made significant progress in the last few decades. Nevertheless, challenges imposed by the immune system remain. The very high doses of AAV vectors used for some disorders have resulted in serious adverse events (SAEs) or even deaths, demonstrating that AAV vector doses that can safely be injected into patients are limited and for some indications below the therapeutic dose. Currently used immunosuppressive drugs have not prevented the SAEs, indicating that it may be prudent to treat patients with repeated transfer of moderate doses rather than a single injection of high doses of AAV vectors. The former approach has been avoided as AAV vectors elicit neutralizing antibodies that prevent successful reapplication of serologically crossreactive vectors. Immunosuppressive regimens that block B cell responses to AAV vectors or treatments that remove AAV neutralizing antibodies thus need to be developed to allow for a shift from toxic single-dose injections of AAV vectors to repeated treatments with more moderate and safe doses. Preventing or blocking antibody responses would also allow for redosing of patients with declining transgene product expression, or for effective AAV-mediated gene transfer into patients with the pre-existing neutralizing antibodies.

INTRODUCTION

The immune system protects an individual from invasions by foreign and potentially damaging entities such as viruses. This in turn puts it at odds with gene therapists, who aim to replace missing or faulty genes with the help of viral vectors. Adeno-associated viruses (AAVs) are commonly used for gene delivery as they are apathogenic, induce only moderate inflammatory responses, infect a wide range of quiescent and dividing cells, and multiple serotypes with distinct tropism are available to preferentially target specific tissues.¹

But like every virus AAVs or AAV-based vectors are immunogenic especially if given at high doses and trigger innate immune responses,² which promote activation of AAV capsid-specific B and T cells.^3,4 AAVs are highly prevalent and infect most humans during infancy.^5,6 Most humans thus have immunological memory to AAV, and many have neutralizing antibodies. As a rule, memory B and T cells are triggered more readily than naïve lymphocytes, and it is more difficult to suppress their activation.⁷

AAV-specific immune cells can negate the benefits of gene therapy; T lymphocytes kill cells that serve as factories for therapeutic proteins,^8,9 while AAV neutralizing antibodies prevent AAV transduction.^10,11 Procedures to allow for effective AAV gene transfer into seropositive patients would not only benefit those with pre-existing immunity, but may also allow for redosing of patients, who achieved only transient or insufficient expression of the therapeutic proteins after the initial gene transfer.¹² It might also allow one to change very high-dose protocols that have resulted in serious adverse events (SAEs), or even death of the same individuals^13

–17 into regimens that give lower and better tolerated doses repeatedly.

Either is only feasible if AAV-specific B cell responses and neutralizing antibodies can be circumvented or suppressed as is addressed in this chapter.

B cell stimulation and recall

Humans carry three distinct subsets of B cells: B1 B cells, marginal zone (MZ) B cells, and follicular B cells; the latter two participate in responses against viruses or viral vectors. MZ B cells could be considered as being part of the innate immune system; they transport antigen and present antigenic peptides to T cells. They can rapidly without T cell help differentiate into plasma cells that fail to undergo affinity maturation and produce mainly antibodies of the IgM isotype.

Follicular B cells, from here on referred to as B cells, are the main B cell subset. They are derived from bone marrow precursors, and circulate in blood or reside in lymphatic tissues. Their activation in general depends on T cell help, and they mature into antibody-secreting plasma cells mainly within germinal centers (GCs) that form in lymphatic tissues upon antigenic challenge. B cells respond to antigen after its binding to the B cell receptor (BCR), a cell surface expressed immunoglobulin (Ig) molecule, which are typically an IgM on naïve B cells and an IgG on memory B cells.

Other molecules on the B cell surface can amplify this signal, most notably CD21, a receptor for complement, which is expressed on all mature B cells. Some antigens, including AAV capsid proteins, bind to complement (C)3 breakdown products such as iC3b,¹⁸ which can in turn bind to CD21. This brings the antigen into proximity to the BCR and through lipid raft formation delays its internalization, resulting in an up to 10,000-fold amplification of BCR signaling.¹⁹

Upon interaction with T helper cells, B cells enter GCs, which are divided into a light and a dark zone. Within the light zone, B cells retrieve antigen from follicular dendritic cells (FDCs). These cells capture antigens through fragment crystallizing (Fc) or complement receptors from macrophages or MZ B cells, and then store them intracellularly in nondegrading compartments from where some of them are periodically recycled to the cell surface. This provides a safe shelter for antigens, which can persist in lymphatic tissues for months or even years.²⁰

The long survival of antigens in turn poses challenges for gene therapist as drugs that suppress B cell activation must be administered for a long time to prevent late B cell activation due to residual immunogens within FDCs. Once B cells capture antigen, it is internalized, degraded, and presented on major histocompatibility (MHC) class II antigens to follicular T helper cells, which in turn provide B cells with survival signals causing upregulation of the transcription factors B-cell lymphoma (BcL)6 and interferon regulatory factor4. This allows B cells to move into the GCs' dark zone where they proliferate, undergo class switching and affinity maturation through activation-induced cytidine deaminase (AID), which through several mechanisms, such as deamination of the cytosine base, causes mutations and DNA breaks in replicating cells.

B cells then return to the light zone where they will compete for antigen carried on the FDCs' surface. B cells that fail to acquire antigen will die while those that express a BCR with competitive affinity will again take up antigen and upon its processing receive survival signals from follicular T helper cells. B cells can then either return to the dark zone for an additional round of proliferation and affinity maturation, or they can exit the GCs as memory B cells, which are typically formed first with the help of the transcription factor broad complex-tramtrack-bric a brac and Cap‘n'collar homology (BACH)2, or antibody-secreting plasma cells.

The latter pathway is promoted by B-lymphocyte–induced maturation protein1, which is in turn inhibited by BcL6 and Forkhead box protein O1, which maintain the B cells' germinal center (GC) program and force them to traffic back to the GC's dark zone. B cells during their differentiation within the hypoxic microenvironment of lymph nodes also upregulate hypoxia-induced factor (HIF)-1α, which regulates B cell migration, metabolism, class-switch reactions, and hypermutation.²¹ Highly repetitive antigens as those on a viral capsid can drive maturation of B cells into plasma cells without T cell help.

As shown previously with antibodies to vesicular stomatitis, virus T cell-independent B cell activation requires that the antigen is present within an ordered structure at a distance of ∼5–10 nm between identical B cell epitopes to allow for crosslinkage of the BCR.²² Nevertheless, T cell-independent activation of B cells even within GCs fails to result in production of affinity-matured antibodies.²³

Plasma cells are divided into long-lived and short-lived plasma cells. The formers are mainly produced within GCs, and they can survive within specialized bone marrow niches and produce antibodies for several months or even the life span of an individual. The latter survive for a few days and are in general activated outside GCs. Memory B cells do not produce antibodies. Upon re-encounter of their cognate antigen, they can rapidly without T cell help differentiate into short-lived plasma cells, or they can enter GCs and upon cycles of antigen capture, T cell help followed by proliferation and affinity maturation, and potentially further class-switching differentiate into new memory B cells or long-lived plasma cells.

Fate decisions of B cells are largely determined by the BCR's signaling strength and by signals from other B cell surface markers such as CD21, which is complexed to CD19 and CD81 or CD40; the latter interacts with CD40 ligand (L) on T helper cells. Molecules downstream of these cell surface signaling molecules offer potential targets to block B cell activation.

Crosslinkage of the BCR through antigen activates spleen tyrosine kinase (SYK) and then Bruton's tyrosine kinase (BTK), which through additional steps leads to induction of the p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (Erk)1/2, and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathways. Although BTK is essential for activation of IgM⁺ B cells, it can be circumvented upon signaling through IgG. Signaling through the CD21-CD19-CD81 complex activates PI3Kδ, and from there the mammalian target of rapamycin (mTOR)/protein kinase B (AKT) pathways. CD40–CD40L interactions induce several tumor-necrosis factor receptor–associated factor adaptor proteins that activate phosphoinositide 3-kinases (PI3K)/AKT.

B cell surface proteins that change during differentiation can be targeted by cell-depleting antibodies. All B cells, but for a very small subset of plasma cells, express CD19. Mature naïve B cells, MZ, GC, and memory B cells are positive for CD20, which is not expressed by plasma cells. GC B cells and plasma cells express CD38, and the latter are also positive for CD138. CD22 is carried by naïve, memory, and GC B cells.

The receptor for B-cell activating factor (BAFF), which regulates cell survival, protein synthesis, and metabolism, is carried by naive and GC B cells. BAFF in addition binds to B-cell maturation antigen (BCMA), which regulates B cell survival and proliferation, and transmembrane activator and calcium-modulator and cyclophilin ligand (CAML) interactor (transmembrane activator and CAML interactor) involved in B cell differentiation; both are expressed on memory B cells, plasmablasts, and plasma cells, and BCMA is also carried by GC B cells.

Naïve B cells upon their stimulation against AAV capsid antigen initially generate antibodies of the IgM isotype and then rapidly switch to production of IgG isotypes. IgG antibodies are composed of two identical light and heavy chains that are linked by disulfide bonds forming a Y-like structure. The N-termini of the four chains form the variable regions that bind the antigen; each arm of an Ig molecule carries the same variable region, which allows for crosslinkage of antigens.

The C-terminus comprises the constant region of an antibody, which defines its isotype (IgM, IgG, IgA, and IgE) and subtype (IgG1-4), and determines the antibody's functions. Plasma cells produce copious amounts of antibodies and can secrete up to 2,000 molecules per second, which poses considerable stress on the cells' secretory pathway, resulting in activation of an unfolded protein response that ensures that protein synthesis, autophagy, and apoptotic signals in case of excessive stress are well coordinated.²⁴ Production of antibodies also requires a supercharged anabolic metabolism that is mainly maintained by amino acids fueling mitochondrial energy production through oxidative phosphorylation, while glucose serves for the glycosylation of antibodies.

Removal of pre-existing AAV neutralizing antibodies

Antibodies can be removed from the blood stream by plasmapheresis. In this process, plasma is collected from the body, and reinfused once large molecules have been removed by centrifugation or filtration. This process is relatively inefficient, and even five rounds fail to eliminate high levels of AAV neutralizing antibodies.²⁵ In addition, plasmapheresis is not selective and removes all antibodies, thus potentially increasing the treated individuals' susceptibility to infections.

A related but more specific approach is hemapheresis with immunoadsorption, which removes antibodies of a selected isotype, or even specifically eliminates AAV capsid-specific antibodies. Removal of IgG, IgA, and IgM antibodies, which is sometimes used to treat autoimmunity or transplant rejection, was shown to reduce or even eliminate antibodies to AAV2 or 5 after three to five rounds of treatment.²⁶

Immunoadsorption of all antibodies of a given isotype or specifically AAV-specific antibodies was shown in experimental animals to reduce AAV neutralizing antibodies to levels that allowed for efficient AAV vector delivery.^27,28 The caveat should be pointed out that eliminating antibodies by either of the above-described methods will only allow for AAV-mediated gene transfer for a relatively short time as AAV-specific plasma cells will eventually replenish the depleted antibodies.

Alternatively, antibodies can be destroyed by enzymes. For example, a cysteine protease from Streptococcus pyogenes called Imlifidase (IdeS) cleaves IgG into Fc and F(ab)₂ fragments. When tested in healthy adults IdeS was shown to eliminate 95% of circulating IgG within 2–4 h.²⁹ IdeS as well as IdeZ, an enzyme with similar functions isolated from S. equi ssp. Zooepidemicus, were shown to allow for AAV transduction of nonhuman primates that had been transfused with human sera containing AAV-specific neutralizing antibodies^30,31; several companies are currently gearing up to explore IdeS for pretreatment of AAV-seropositive gene therapy recipients.^32,33

Although F(ab)₂ fragments can still neutralize virus, they have a shortened half-life and are rapidly eliminated. Another approach based on the same principle, that is, to eliminate antibodies by accelerating their degradation, has been to block neonatal Fc receptor (nFcR), which extends the half-life of IgG by preventing its lysosomal digestion once the antibody is taken up by a cell. An antibody that blocks the interactions between IgG and nFcR was shown to reduce AAV neutralizing antibodies three- to fourfold, thereby allowing for transduction upon intravenous AAV gene transfer into seropositive animals.³⁴

In yet another approach that was first tested in animals and then in hemophilia patients undergoing AAV-mediated factor replacement therapy, the vectors encoding the therapeutic protein were mixed with an excess of so-called “empties,” that is, AAV shells without genomes, under the expectation that the empties would soak up the neutralizing antibodies. This method was shown to effectively circumvent low titers of neutralizing antibodies in animals. Adding empties comes at a price. In mice with immunological memory to AAV as well as in humans empty AAV particles trigger CD8⁺ T cell recall responses,^35,36 which can eliminate the AAV vector-transduced cells⁸ (Table 1).

Table 1.

Removal of antibodies

Drug	Name	Mode of action	Target	FDA-approved indications	Pediatric populations	Potential side effects
AAV empties	—	Removes AAV neutralizing antibodies	AAV-specific antibodies	N/A	—	Increased activation of AAV capsid-specific T cells
Plasmapheresis	—	Removes large molecules from plasma	Unspecific	Myasthenia gravis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy	—	Hypotension, hypocalcemia, hypomagnesemia
Immunoadsorption	—	Removes specific IgG isotypes or even only AAV-specific antibodies	Isotype or antigen specific	Autoimmunity, antibody-mediated transplant rejection	—	Dizziness, nausea
Imlifidase	Idefirix	Degrades IgG into F(ab’)2 and Fc fragment with short half-life	IgG antibodies	Desensitization of kidney transplant recipients (EU/UK)	Safety and efficacy not established in children	Infections
Efgartigimod alfa	Vyvgart	Blocks binding of IgG to the nFcR thereby causing its intracellular degradation	IgG antibodies	Myasthenia gravis	Safety and efficacy not established in children	Infections, hypersensitivity reactions

AAV, adeno-associated virus; Fc, fragment crystallizing; FDA, Food and Drug Administration; N/A, not applicable; nFcR, neonatal Fc receptor.

Circumventing antibodies by using different AAV serotypes

Neutralizing antibodies against AAV capsid antigen are highly crossreactive, thus limiting the availability of serotypes that escape neutralization by antibodies induced by a natural infection or prior AAV gene transfer. Using AAV vectors based on different serotypes or vectors with artificial capsids from which neutralizing epitopes had been removed showed some promise in mice,^37,38 but may be impractical for clinical applications as it would require the very costly GMP production and release testing of not just one but two different AAV vectors. In addition, the B cell repertoire is in part defined by the genetic makeup of an individual, so that presence of B cells able to produce AAV-crossreactive antibodies will vary between patients.

Eliminating B cells by antibody-mediated lysis

B cell subsets can be removed by infusion of antibodies that cause their destruction—a process that is used routinely for treatment of B cell malignancies or antibody-mediated autoimmune diseases. CD20 is expressed on most B cells except plasma cells and targeted by rituximab, a Food and Drug Administration (FDA)-approved monoclonal antibody. Rituximab combined with other immunosuppressive drugs has been tested in experimental animals for vector redosing.³⁹

One clinical trial treated infants with Tay-Sachs disease with an AAV8 vector given together with rituximab, sirolimus, and steroids.⁴⁰ In a recently completed trial, patients with late onset Pompe disease were treated with rituximab and sirolimus before injection of an AAV9 vector for alpha-glucosidase, which was given twice at a 4 months interval.⁴¹ Results from this trial are not yet available.

CD19 is expressed on nearly all B cells including most plasma cells; antibodies against CD19 such as Zynlonta are being used for treatment of B cell cancers. Although antibodies against CD19 have an extended breadth compared with those against CD20 by also depleting plasma cells, expression levels of the former are comparatively low. Other antibodies that are being developed for autoimmune diseases and that could be explored to block antibody responses to AAV gene transfer have a narrower range of targets as shown in Table 2.⁴²

Table 2.

Removal of B cells

Antibody, drug	Name	Mode of actions	Target						FDA-approved indications	Pediatric populations	Potential side effects
Antibody, drug	Name	Mode of actions	FDA approved	Naïve B cells	GC B cells	Memory B cells	Plasma blasts	Plasma cells	FDA-approved indications	Pediatric populations	Potential side effects
Anti CD19	Zynlonta	Antibody-mediated lysis of B cell subsets	Yes	Yes	Yes	Yes	Yes	Yes	B cell lymphoma	Safety and efficacy not established in children	Tiredness, nausea, diarrhea or constipation, abdominal pain, vomiting, decreased appetite, or muscle/joint pain
Anti-CD20	Rituximab		Yes	Yes	Yes	Yes	Yes	No	CD20-positive B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, microscopic polyangiitis, and granulomatosis with polyangiitis, pemphigus vulgaris	6 months and older	Bleeding, aches, chills
Anti-CD38	Daratumumab		Yes	No	Yes	No	Yes	Yes	Multiple myeloma, relapsed/refractory T-cell acute lymphoblastic leukemia, lymphoblastic lymphoma	Tested in children >1 year of age	Hypoxia, dyspnea, hypertension, tachycardia, bronchospasm, anaphylactic reaction
Anti-CD22	Inotuzumab ozogamicin		Yes	Yes	Yes	Yes	No	No	Relapsed or refractory B-cell precursor acute lymphoblastic leukemia	Tested in children >1 year of age	Thrombocytopenia, neutropenia, infection, anemia, leukopenia, fatigue, hemorrhage, pyrexia, nausea, headache, transaminitis, abdominal pain
Anti-BAFF-R	Belimumab		Yes	Yes	Yes	No	No	No	Systemic lupus erythematosus	Approved for children 5–17 years of age	Bleeding, pain, chills, cough, diarrhea, fever, nausea, dizziness
Anti-BCMA	Belantamab mafodotin		Yes	No	Yes	Yes	Yes	Yes	Relapsed or refractory multiple myeloma	Safety and efficacy not established in children	Constipation, diarrhea, nausea, fatigue
Anti-APRIL (TACI ligand)	BION1303		No	No	No	Yes	Yes	Yes	Investigational drug for IgA nephropathy	—	—
Bortezomib	Velcade	Inhibition of proteasomes, apoptosis due to ER stress	Yes	No	No	No	Yes	Yes	Multiple melanoma, mantle cell lymphoma	Safety and efficacy not established in children	Decreased blood clotting, liver damage, thrombotic microangiopathy

APRIL, A Proliferation Inducing Ligand; BAFF-R, B-cell activating factor receptor; BCMA, B-cell maturation antigen; ER, endoplasmic reticulum; GC, germinal center; TACI, transmembrane activator and CAML interactor.

Eliminating B cells by blocking protein degradation

The high anabolic activity of B cells, whose job it is to churn out antibody molecules by the thousands, requires a well-functioning proteolytic machinery that can eliminate misfolded proteins. Interestingly though, while the size of the endoplasmic reticulum increases markedly upon differentiation of B cells into antibody-secreting plasma cells, numbers of proteasomes decline, which puts the cells at an enhanced risk for apoptotic cell death due to accumulation of ubiquitinated proteins. It also makes plasma cells susceptible to further inhibition of proteasome activity such as through the drug bortezomib, which causes their cell cycle arrest and apoptosis.

Brotezomib has a boronic group that binds to a threonine hydroxyl group on the ß5 unit of proteasomes, thereby blocking its enzymatic activity. Brotezomib has been shown to reduce levels of pre-existing AAV-specific antibodies in mice, although levels remained too high to permit effective redosing on an AAV vector.⁴³ More promising results were obtained in mice upon combining Bortezomib with an antibody to CD20.⁴⁴ Bortezomib has another benefit for AAV-mediated gene transfer; it enhances AAV vector transduction rates⁴⁵ and decreases presentation of capsid-derived peptides by MHC class I molecules, thus potentially reducing activation and effector functions of CD8⁺ T cell responses⁴⁶ (Table 3).

Table 3.

Inhibition of B cell activation

Drug	Name	Mode of action	Target	FDA-approved indications	Pediatric populations	Potential side effects
Bortezomib	Velcade	Inhibits proteasomes, apoptosis due to ER stress	Plasma cells	Multiple melanoma, mantle cell lymphoma	Safety and efficacy not established in children	Bleeding, liver damage, thrombotic microangiopathy
Azathioprine	Azasan	Purin metabolism antagonist	Proliferating B cells	Transplant rejection, rheumatoid arthritis, inflammatory bowl disease, hepatitis	Used in children	Fever, rash, weight loss, dizziness
Mycophenolate mofetil	CellCept	Depletes guanosine nucleotides	Proliferating B cells	Transplant rejection	Used in children ≥3 months of age	Fever, allergic reactions, bleeding, fatigue
Cyclosporine	Sandimmune, Neoral, Gengraf	Blocks production of cytokines, inhibits B cell proliferation, destabilizes HIF-1α	T cells, GC B cells	Transplant rejection, rheumatoid arthritis, autoimmunity	Used in children	Liver and kidney problems, infection, and high blood pressure
Rapamycin	Sirolimus, Rapamun	Inhibits activation of mTOR	GC B cells, MZ	Transplant rejection, lymphangioleiomyom, additional orphan deignationsatosis	Used in children	Sore and inflamed mouth, weakened immune system, anemia, fatigue, high blood pressure, constipation, or diarrhea
Ibrutinib	Imbruvica	Inhibits BTK signaling	GC B cells	Chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström's macroglobulinemia	Used in children ≥1 year of age	Bleeding, blurred vision, rash, chills, confusion, cough, nausea, headaches, seizures, vomiting
APL-2	Pegcetacoplan	Blocks C3 activation	GC B cells	Paroxysmal nocturnal hemoglobinuria	Ages 12–17, study ongoing	Bleeding, swelling, blurred vision, chills, confusion, flu-like symptoms, seizures, weight gain

BTK, Bruton's tyrosine kinase; HIF-1, hypoxia-induced factor-1; MZ, marginal zone; mTOR, mammalian target of rapamycin.

Drugs that target B cell differentiation

Several immunosuppressive drugs target B cell differentiation pathways. Steroids, which are commonly used to block destruction of AAV-transduced cells by AAV-capsid or transgene product-specific CD8⁺ T cells,⁴⁷ inhibit plasma cell differentiation by reducing expression of Blimp and BcL6.⁴⁸ Nevertheless, AAV gene transfer trials that upon using steroids such as prednisolone reported a reduction in CD8⁺ T cell responses also showed that antibody responses to AAV capsid remained unaffected.

Azathioprine (AZA) is an antagonist of purine metabolism, resulting in the inhibition of DNA, RNA, and protein synthesis, thereby reducing numbers of circulating B and T cells. It can also eliminate T cells undergoing activation by subverting Rac family small GTPase (Rac)1 induction upon CD28 signaling, resulting in apoptosis, thereby lacking T cell help for B cells.⁴⁹

Mycophenolate mofetil (MMF, CellCept^®) is a prodrug of mycophenolic acid and has a similar activity by inhibiting inosine monophosphate dehydrogenase, an enzyme needed for the de novo synthesis of guanosine nucleotides. AZA or MMF has been used in clinical trials in combination with tacrolimus, a calcineurin inhibitor, to stop T cell responses upon transfer of an AAV vector for factor VIII.⁴⁷ MMF was also used with thymocyte globulin (ATG) and cyclosporine in dogs as described below.

Cyclosporine has been used in combination with a nondepleting antibody to CD4 to dampen antibody responses to AAV gene transfer in mice and to allow for a second administration of the same vector.⁵⁰ In another study, cyclosporin combined with MMF allowed for sustained transgene product expression after transfer of an AAV vector expressing microdystrophin into the legs of dystrophic dogs. Cyclosporin inhibits B cell proliferation, and by destabilizing HIF-1 α affects B cell migration. In addition, cyclosporin blocks T cell help by preventing calcineurin-dependent dephosphorylation of the transcription factor nuclear factor of activated T cells, thereby producing cytokines and chemokines such as interleukin-2.

Rapamycin has mainly been explored preclinically for inhibition of AAV-induced B cell responses. In its native form, it was only modestly effective, while upon its delivery within poly(lactic-co-glycolic acid), preglycated polylactic acid nanoparticles, it was shown to inhibit T and B cell activation in mice and nonhuman primates, thereby allowing for readministration of an AAV vector.

In mice, the effect was transferrable by CD25⁺ splenocytes, suggesting a contribution of regulatory T cells (Tregs).⁵¹ Rapamycin, also known as Sirolimus, inhibits mTOR signaling, which is downstream of the BCR/CD19 pathway and is essential to increase protein synthesis, metabolism, and antiapoptotic pathways in B cells that are in the process of being activated. Rapamycin also inhibits BAFF-R signaling, reduces BcL6 and AID expression, and thereby prevents B cell differentiation into plasma or memory B cells.⁵²

BTK is key signaling molecule downstream of the BCR. Crosslinkage of the BCR induces proto-oncogene tyrosine-protein kinase (Src) family members Lck/Yes novel tyrosine kinase (Lyn) and spleen-associated tyrosine kinase (Syk) to phosphorylate BTK at tyrosine (Tyr)₅₅₁, which then increases its catalytic activity further by autophosphorylation at Tyr₂₂₃.

Ibrutinib binds to the catalytic site of BTK at cysteine (Cys)₄₈₁ of BTK and further inhibits autophosphorylation of Tyr₂₂₃, thereby blocking its enzymatic activity. In mice ibrutinib given together with rapamycin before and during AAV gene transfer inhibited primary anti-AAV capsid-specific antibody responses, slightly reduced recall responses, but failed to prevent formation of specific memory B cells. Either drug given alone was ineffective.⁵³

Inhibiting binding of complement components to AAV capsid

As described above, AAV capsid antigens can bind C3 breakdown products, and thereby upon binding to CD21 amplify BCR signaling. Previous studies showed that antibody responses to AAV capsid are reduced (5–10-fold) but not abolished in C3 knockout mice.¹⁸ C3 inhibition by drugs such as APL-2 or APL-9, two peptides that upon binding to C3 and C3b inhibit their further activation and thereby block all three pathways of complement stimulation,⁵⁴ would be expected to moderate and shorted Ab responses to AAV by reducing the amount of antigen on FDCs and B cells. This in turn might enhance the inhibitory effects of drugs that target B cell signaling. Alternatively, one could attempt to identify and eliminate the C3 binding site on AAV capsid.

Inducing Tregs response

Tregs can very efficiently inhibit adaptive T and B cell responses, and they have been shown to play a role in preventing transgene product-specific immune responses upon AAV gene transfer.⁵⁵ Others have shown that the immunosuppressive effects of rapamycin in part relate to its effect on cell metabolism that favors inductions of Tregs over stimulation of effector T cell subsets.⁵⁶

It has been proposed that in vitro-generated or expanded Tregs could facilitate tolerance to AAV gene transfer. One study showed that AAV-CAR Tregs based on T cells with a chimeric AAV-specific antibody receptor linked to costimulators and forkhead box P (FoxP)3 could indeed suppress T cell responses to AAV capsid as well as antibody and T cell responses to the transgene product but failed to reduce antibody responses to the AAV capsid.⁵⁷

The idea of inhibiting destructive immune responses to AAV by inducing Tregs is attractive; notwithstanding, evolution most likely ensured that Tregs cannot effectively suppress the redundancy of pathways that allow for activation of antiviral antibodies. Ultimately antibodies are the immune system's most crucial elements to ensure survival of our species as best shown by the need for their maternal transfer.

CONCLUSIONS

AAV-mediated gene transfer has tremendous potential to cure monogenic disorders. Although gene transfer with moderate doses of AAV vectors is well tolerated, the very high doses that are needed to correct many diseases, especially those that require treatment during infancy or early childhood, have led to SAEs and deaths even in patients that were concomitantly treated with immunosuppressive drugs. One potential approach to allow for high doses of AAV vectors without unacceptable risk would be the repeated transfer of lower vector doses. Other patients lose transgene product expression over time and would benefit from a second AAV gene transfer.

Repeated AAV gene transfer is problematic as AAV vectors induce very potent capsid-specific neutralizing antibody responses that impede the success of additional gene transfer using the same or a serologically crossreactive vector. In addition, many individuals have pre-existing AAV neutralizing antibodies due to a natural infection, which precludes them from AAV-mediated gene therapy.

Several approaches have been tried preclinically or clinically to either remove circulating AAV neutralizing antibodies or block induction of B cell response upon AAV-mediated gene transfer. Although some studies reported successes especially in animals that had no prior encounter with AAV antigens, complete long-term removal of high levels of AAV neutralizing antibodies or inhibition of memory B cell responses will continue to be a challenge that most likely will require combination of drugs that may need to be given for an extended period.

Footnotes

ACKNOWLEDGMENTS

I thank C. Cole for assistance with editing the article.

AUTHOR'S CONTRIBUTION

H.C.J.E. is solely responsible for the drafting and editing of this article.

AUTHOR DISCLOSURE

H.C.J.E. is a cofounder of Virion Therapeutics, Inc. She serves as a consultant to several gene therapy companies.

FUNDING INFORMATION

Virion Therapeutics (52071-01-307), the Wellcome Trust, and the Wistar Science Discovery Fund (10000-01-307).

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