Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses

Ocular AAV treatments have caused permanent visual acuity loss after intraocular inflammation.

“ocular AAV therapies leading to a permanent reduction in visual acuity following intraocular inflammation (9–11)”

• cites [9] sourcePartly supported

  The Results (Inflammatory and Immune Responses; page unavailable) report that mild anterior uveitis in one participant was followed by a persistent 15-letter reduction in visual acuity. This supports a lasting loss after intraocular inflammation, but the paper reports persistence during follow-up, not that the loss was permanent or irreversible. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [10] sourceNot checked — no full text of the source
• cites [11] sourcePartly supported

  The Results report significant retinal inflammation in patient C2 two weeks after AAV treatment, and Online Methods describe it as visually significant vitritis and retinitis. Neither passage establishes a permanent reduction in visual acuity. The Discussion also says the inflammation might have been related to the injection surgery, so its cause is uncertain. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

In mice, 1e11 vg scAAV8.FIX given systemically caused moderate liver type I interferon gene expression at 2 hpi, the peak time.

“systemic administration of 1 × 1011 vector genomes (vg) (equivalent to ~4 × 1012 vg/kg, a therapeutically relevant dose in the clinic) of scAAV8.FIX in mice stimulated moderate type I interferon (Ifnb1 and Ifna13) gene expression in the liver at 2 hours post-injection (hpi), the peak time point as previously described (16).”

• cites [16] sourcePartly supported

  The Gene expression comparison section reports hepatic IFN-α/β expression after 1 × 10^11 vg of scAAV2-F.IX in mice, with responses declining after the 2-hour measurement. Figure 1 and the accompanying text identify the tested scAAV8 vector as scAAV8-GFP, not scAAV8.FIX; the time course shown is for scAAV2-F.IX. Thus the source supports the dose, liver response, and 2-hour timing, but not the claim that these interferon results or the peak time were demonstrated specifically for scAAV8.FIX. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The AAV vector genome triggers TLR9, a receptor that detects foreign DNA.

“the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA”

• Also stated in: § INTRODUCTION · boosted

  “Several studies have demonstrated a central role for Toll-like receptor 9 (TLR9), an immune sensor of DNA, in detecting AAV genomes and triggering innate immune and CD8+ T cell responses (15–18).”

• cites [15] sourceSupported
• cites [16] sourceSupported
• cites [17] sourceSupported
• cites [18] sourceSupported

• AssumptionRests on an assumption: CpG motifs are present in AAV vector genomes.

Clinical trials have reported AAV dose-dependent intraocular inflammation and persistent visual acuity loss after subretinal AAV despite prophylactic immunosuppression.

“AAV dose-dependent intraocular inflammation and a few instances of persistent loss of visual acuity have been independently reported in clinical trials following subretinal AAV administration despite prophylactic immunosuppression (9–11).”

• cites [9] sourcePartly supported

  The Results (Inflammatory and Immune Responses; page not provided) report inflammation in higher-dose recipients and a persistent 15-letter loss of visual acuity in one participant. The Adverse Events section mentions oral glucocorticoids, but the passages in our copy do not establish that immunosuppression was given prophylactically. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [10] sourceNot checked — no full text of the source
• cites [11] sourcePartly supported

  The Online Methods report visually significant vitritis and retinitis in patient C2 despite prophylactic prednisolone. The Results identify C2 as a high-dose recipient, but neither the Results nor Discussion establishes a dose-dependent pattern of inflammation. They also do not report persistent visual-acuity loss; the Results instead report an overall improvement in treated eyes at two years. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

The innate response to self-complementary AAV in mice required TLR9/MyD88.

“We confirmed with Tlr9−/− and Myd88−/− mice that the innate immune response to self-complementary AAV was indeed dependent on the TLR9/MyD88 pathway (Fig. 1D and 1E), as previously shown in the literature (15–17).”

• cites [15] sourcePartly supported

  The research article (p. 3) reports that AAV2-induced type I interferon production by mouse plasmacytoid dendritic cells depended on MyD88, and Figure 5 (p. 4) reports no detectable response in Tlr9−/− or Myd88−/− cells. Our copy does not identify the tested vectors as self-complementary AAV, so it does not establish that qualifier. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [16] sourceSupported
• cites [17] sourcePartly supported

  The Results (page unavailable) show that Tlr9-deficient mice had a reduced innate cytokine and chemokine response to AAVrh32.33. The Introduction and Discussion (pages unavailable) connect TLR9 to self-complementary AAV and report TLR9/MyD88-dependent recognition of AAV2, but they do not establish that the innate response to self-complementary AAV in mice required both TLR9 and MyD88. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Prior NHP intravitreal AAV work found that increasing the dose 10-fold produced only a 2-fold increase in aflibercept expression.

“in line with a previous intravitreal AAV study in NHPs showing that a 10-fold higher AAV dose only resulted in 2-fold more aflibercept expression (34).”

• cites [34] sourceSupported

CD8+ T cell responses against rh32.33 capsid required MyD88 in mice.

“We confirmed with Myd88−/− mice that development of CD8+ T cell responses against rh32.33 capsid depended on MyD88(15, 17) (Fig. 2A, middle panel).”

• cites [15] sourcePartly supported

  The CD8+ T-cell results (p. 4, Figure 6C–D) show significantly diminished capsid-specific responses in Myd88-deficient mice. But the tested capsid was AAV2, not rh32.33; our copy does not establish the claim for that capsid. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [17] sourcePartly supported

  The Results and Figure 2 caption (page unavailable) report reduced capsid-reactive CD8+ T-cell responses in Tlr9-deficient mice. They do not report an experiment in Myd88-deficient mice or establish MyD88 dependence for the rh32.33 capsid response; the Discussion mentions MyD88 only when describing another study’s broader AAV findings. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Intravitreal AAV delivery is known to pose greater immunogenicity challenges than subretinal delivery.

“The immunogenicity challenges of intravitreal administration of AAV are well-recognized compared to subretinal administration (35).”

• cites [35] sourceSupported

Putting the TLR9i sequence at the 5' end also reduced NF-κB and IL-8.

“When the order of ODN 2006 and the covalently linked TLR9i sequence was reversed so that the TLR9i sequence was at the 5’ end, a similar reduction in NF-κB response, as well as a reduction in the pro-inflammatory cytokine IL-8, were observed”

ODN TTAGGG did not inhibit TLR7 or TLR2/6 activation even at high concentration.

“ODN TTAGGG, a well-known TLR9i sequence derived from mammalian telomeres (23), did not inhibit the activation of TLR7 or TLR2/6 (sensors of microbial RNA and lipoproteins, respectively) even at high concentration”

ODN 2006-TLR9i with ODN 2006 at the 5' end produced a much lower NF-κB response than ODN 2006 linked to control sequences.

“treatment of cells with ODN 2006-TLR9i oligonucleotides (with the ODN 2006 sequence at the 5’ end) resulted in markedly reduced NF-κB response compared to ODN 2006 linked to two separate control sequences not expected to stimulate or inhibit TLR9 (control1 or control2)”

• AssumptionRests on an assumption: The control sequences do not stimulate or inhibit TLR9.

A five-nucleotide AAAAA spacer between ODN 2006 and TLR9i sequences ODN 2088 or TTAGGG gave similar NF-κB reduction.

“Insertion of a 5 nt intervening sequence (AAAAA) between ODN 2006 and selected TLR9i sequences (ODN 2088 or TTAGGG) yielded similar results”

scAAV8.FIX.io1 did not raise interferon gene expression above PBS at 4 hpi.

“scAAV8.FIX.io1 treatment also did not elevate interferon gene expression compared to PBS treatment 4 hpi (fig. S2).”

The same dose of scAAV8.FIX.io1 caused no detectable innate immune response in mouse liver.

“the same dose of scAAV8.FIX.io1 elicited no detectable innate immune response in the liver (Fig. 1B).”

At 1e11 vg, both rh32.33.GFP and rh32.33.GFP.io2 caused very high capsid-directed T cell responses (~800-1000 SFU/million splenocytes).

“At a higher dose of 1 × 1011 vg, both rh32.33.GFP and rh32.33.GFP.io2 elicited extremely high capsid-directed T cell responses (~800 – 1000 SFU/million splenocytes) (Fig. 2A, right panel).”

At 5 × 10^11 vg in NHPs, AAV2.aflibercept.io2 did not lower the rate of clinical uveitis.

“While AAV2.aflibercept.io2 did not reduce the incidence of clinical uveitis at 5 × 1011 vg (Fig. 5A; compare Group 4 to 5 and Group 6 to 7)”

In intravenous scAAV8.FIX experiments, IFN-γ ELISpot detected no appreciable CD8+ T cell response against AAV8 capsid.

“in our intravenous scAAV8.FIX experiments, we did not detect appreciable CD8+ T cell responses against the AAV8 capsid in an IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (fig. S4).”

Prophylactic systemic steroids did not boost aflibercept expression in the NHP intravitreal study.

“The use of prophylactic systemic steroids did not boost aflibercept expression (Fig. 5B, compare Groups 6 and 7 to Groups 4 and 5).”

io2 incorporation produced earlier, more extensive, and longer-sustained GFP expression in mouse retina in vivo.

“incorporation of io2 resulted in earlier, more extensive and greater sustained expression of GFP transgene in vivo observable by longitudinal intravital retinal imaging (Fig. 3C and fig. S10A).”

Prophylactic systemic steroids did not reduce the incidence of clinical uveitis in the NHP intravitreal study.

“Use of prophylactic systemic steroids similarly failed to reduce the incidence of clinical uveitis”

When no innate response occurred, scAAV8.FIX.io1 did not increase FIX expression over scAAV8.FIX.

“In experimental conditions where an innate immune response was not triggered (administration of 10-fold lower doses of AAV or administration to Myd88−/− mice), scAAV8.FIX.io1 did not lead to an enhancement in FIX expression (Fig. 1H and 1I)”

At 6 weeks, AAV8.GFP caused marked loss, shortening, or altered morphology of cone outer segments in all five injected outbred pigs.

“at 6 weeks post-injection (wpi), in all five AAV-injected outbred pigs, AAV8.GFP led to a marked loss, shortening or altered morphology of cone outer segments as labeled by expression of opsins (Fig. 4A and fig. S12A)”

Higher-dose subretinal AAV in pigs caused photoreceptor pathology with microglia and T cell infiltration.

“Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration.”

AAV2.GFP.WPRE.io2 caused lower IL-1β responses than AAV2.GFP.WPRE in pDCs from 9/10 responding donors and moDCs from 9/11 donors.

“AAV2.GFP.WPRE.io2 elicited lower IL-1β cytokine responses than the unmodified vector in pDCs from nine out of ten donors whose cells responded to AAV infection, and in monocyte-derived dendritic cells (moDCs) from nine out of eleven donors (Fig. 2G, fig. S6).”

In pig animals 23585 and 23586, elevated numbers of CD8+ T cells infiltrated the outer and inner retina in eyes that received AAV8.GFP, but not in contralateral eyes that received AAV8.GFP.io2.

“In these same animals, we detected elevated numbers of CD8+ T cells infiltrating the outer and inner retina in the eye that received AAV8.GFP, but not in the contralateral eye receiving AAV8.GFP.io2 (Fig. 4C and fig. S13C).”

scAAV8.FIX.io1 raised human FIX in plasma about threefold over scAAV8.FIX at 28 days.

“scAAV8.FIX.io1 enhanced human FIX in plasma nearly 3-fold compared to scAAV8.FIX 28 days post-injection (dpi) (Fig. 1G)”

In macaques, higher-dose intravitreal AAV with the engineered vector delayed but did not prevent clinical uveitis.

“intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis”

Iba1 signal increased in all AAV-injected pig eyes at 6 weeks after injection.

“Iba1 signal increased in all AAV-injected eyes (Fig. 4B), suggesting microglia activation and proliferation.”

Pig 23585 had severe vitritis at 2 weeks in the AAV8.GFP eye but not in the contralateral AAV8.GFP.io2 eye.

“animal 23585 experienced severe vitritis at 2 wpi in its AAV8.GFP-treated eye, but not the contralateral eye treated with AAV8.GFP.io2 (fig. S16 and table S3).”

At 1e10 vg intramuscularly, rh32.33.GFP caused CD8+ T cell responses against an immunodominant capsid epitope at 21 days in 7 of 10 mice.

“at a dose of 1 × 1010 vg injected into the quadriceps muscle, rh32.33.GFP triggered a range of CD8+ T cell responses against an immunodominant capsid epitope at 21 dpi, with 7 of 10 animals showing positive T cell reactivity (Fig. 2A, left panel).”

Tenfold lower doses of scAAV8.FIX or scAAV8.FIX.io1 did not trigger liver interferon responses.

“Administration of 10-fold lower doses (1 × 1010 vg) of either scAAV8.FIX or scAAV8.FIX.io1 did not trigger interferon responses in the liver”

In the NHP study, prophylactic systemic steroids postponed clinical uveitis by about 22 days for both vectors.

“but we did note a delay by ~22 days for both AAV2.aflibercept and AAV2.aflibercept.io2 (Fig. 5A, compare Group 4 to 6 and Group 5 to 7).”

All injected pig eyes had severe laminar damage around the subretinal injection site, surrounded by non-severe laminar disruption.

“In all injected eyes, there was an area of severe laminar damage surrounding the site of the subretinal injection, which was further surrounded by a region of non-severe laminar disruption (fig. S14).”

At 5 × 10^11 vg, clinical uveitis began latest when io2 and prophylactic systemic steroids were combined.

“The combination of io2 and prophylactic systemic steroids gave the latest onset of clinical uveitis at the 5 × 1011 vg dose (Fig. 5A, Group 7)”

AAV2.GFP.WPRE.io2 reduced ocular T cell numbers compared with AAV2.GFP.WPRE in mice.

“AAV2.GFP.WPRE.io2 reduced ocular T cell numbers compared to AAV2.GFP.WPRE (Fig. 3B, fig. S8B and fig. S9).”

In pig eyes, GFP expression in photoreceptors was not higher in AAV8.GFP.io2-treated eyes than in AAV8.GFP-treated eyes.

“We did not observe enhanced GFP expression in photoreceptors of AAV8.GFP.io2-treated eyes compared to AAV8.GFP (fig. S13C)”

Flow cytometry of dissociated mouse eyes showed robust CD45+ leukocyte infiltrate containing CD3+ and CD8+ T cells.

“We dissociated eye tissues for flow cytometry analysis and surprisingly observed robust CD45+ leukocyte infiltrate, comprising CD3+ T cell and CD8+ T cell populations.”

rh32.33.GFP.io2 muscle sections had higher GFP expression than unmodified vector by immunohistochemistry.

“rh32.33.GFP.io2 muscle sections exhibited higher GFP expression than those with the unmodified vector by immunohistochemical analysis”

These adverse effects were avoided in contralateral eyes injected with engineered vectors.

“These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors.”

In pig eyes at 6 weeks after subretinal injection, un-injected and vehicle-injected eyes showed Iba1 staining with a ramified pattern outside the outer nuclear layer, consistent with resting microglia.

“Iba1 staining of un-injected and vehicle-injected eyes showed a ramified staining pattern outside the outer nuclear layer (ONL), consistent with resting microglia (Fig. 4B).”

In NHP vitreous, average aflibercept expression was about 2-fold higher with AAV2.aflibercept.io2 than with AAV2.aflibercept at both 1 × 10^11 vg and 5 × 10^11 vg.

“However, we did note that AAV2.aflibercept.io2 resulted in an approximately 2-fold enhancement in average aflibercept expression compared to AAV2.aflibercept at both the 1 × 1011 vg and 5 × 1011 vg doses (Fig. 5B, compare Group 2 to 3 and Group 4 to 5).”

Both scAAV8.FIX and scAAV8.FIX.io1 produced similar anti-AAV8 neutralizing antibody titers at 28 days.

“We observed the development of comparable amounts of neutralizing antibody (NAb) titers at 28 dpi against AAV8 for both scAAV8.FIX and scAAV8.FIX.io1”

In two AAV-injected pigs, laminar changes were similar between right and left eyes at 2 weeks.

“In both animals, laminar changes were similar between right (OD) and left (OS) eyes at 2 wpi (fig. S14B, S14C, S15B and S15C).”

In the NHP intravitreal study, aflibercept levels did not show a dose-dependent rise from 1 × 10^11 vg to 5 × 10^11 vg.

“Comparing the 1 × 1011 vg and 5 × 1011 vg groups, there was no evidence of a dose-dependent increase in aflibercept concentrations (Fig. 5B; compare Groups 2 and 3 to Groups 4 and 5)”

In vehicle-injected pig retina, laminar changes at 2 weeks were reduced by 6 weeks.

“In the vehicle-injected retina, the laminar changes evident at 2 wpi were reduced at 6 wpi (fig. S14A and S15A).”

AAV2.GFP.WPRE.io2 caused lower IFN-β responses than AAV2.GFP.WPRE.

“AAV2.GFP.WPRE.io2 also elicited reduced IFN-β cytokine responses compared to AAV2.GFP.WPRE (Fig. 2H).”

GFP transgene expression did not appear to affect overall retinal cell viability.

“expression of the GFP transgene did not appear to impact the overall viability of retinal cells (fig. S10C).”

In several models spanning different capsids, genome designs, promoters, transgenes, tissues, routes, and animals, vectors containing TLR9i sequences markedly reduced immune responses without altering other sequences.

“We directly compared AAV vectors with or without TLR9i sequences in experiments encompassing different capsid serotypes, genome configurations, promoters and transgenes, target tissues, routes of administration and animal models, and showed marked reduction of immune responses without changing any other sequences in several models.”

Including or omitting the extra T in the ODN TTAGGG sequence did not change TLR9-inhibitory activity.

“No differences in TLR9-inhibitory activity were observed with or without the inclusion of the additional T.”

Host immune reactions are a major obstacle to in vivo gene transfer.

“host immune responses pose a major challenge for in vivo gene transfer (1)”

• cites [1] sourceNot checked — no full text of the source

Structural work described two TLR9i sequences that form stem-loop structures fitting inside the TLR9 ring and blocking dimerization and activation.

“Structural studies recently described two different TLR9i sequences that formed stem-loop structures that fit snugly into the interior of the ring structure of TLR9, preventing dimerization and activation (21).”

• cites [21] sourceNot checked — no full text of the source

A TLR9i sequence in the same molecule as inflammatory DNA may dampen inflammation more effectively than separate administration.

“Thus, incorporation of a TLR9i sequence within the same molecule as an inflammatory DNA sequence could be more effective at dampening inflammation than administering it as a separate molecule.”

Deaths have been reported in a high-dose systemic AAV trial for children with X-linked myotubular myopathy.

“deaths have been reported in a high dose systemic AAV gene therapy trial for children with X-linked myotubular myopathy (14)”

• cites [14] sourceNot checked — no full text of the source

TLR9i sequences can still act when placed away from CpG motifs.

“suggesting TLR9i sequences also can function when they are distal to CpG motifs”

ODN TTAGGG is specific for TLR9.

“supporting its specificity for TLR9”

The coupled immunomodulation strategy may widen the therapeutic window for AAV therapies and other DNA-based gene transfer methods.

“This “coupled immunomodulation” strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.”

Immune factors besides TLR9 may contribute to intraocular inflammation in the macaque model.

“suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model.”

io2 may be partly protective against T cell responses at the higher dose.

“suggesting that io2 might be partially protective against T cell responses at the higher dose”

Inflammation seen clinically does not fully reflect the amount of inflammation, especially immune responses confined to retinal tissue.

“implying that clinically observable inflammation incompletely captures the extent of inflammation, particularly tissue-localized immune responses in the retina.”

In the pig retina, the TLR9-inhibitory engineered vector, but not the unmodified vector, may prevent undesirable innate and adaptive immune cell responses.

“Taken together (see summary in table S3), these data strongly suggest that, unlike the unmodified vector, the engineered vector carrying TLR9-inhibitory sequences could avoid eliciting undesirable innate and adaptive immune cell responses in the retina.”

The TLR9-inhibitory approach is valuable for avoiding innate responses in human pDCs.

“These results underscored the value of our approach in evading innate immune responses in human pDCs.”

The effects of io2 and prophylactic systemic steroids on clinical uveitis onset were additive.

“suggesting that the effects of io2 and steroids were additive.”

Linking immunomodulatory non-coding sequences to longer therapeutic nucleic acids can cloak the vector from unwanted immune responses in multiple, but not all, models.

“linking specific immunomodulatory non-coding sequences to much longer therapeutic nucleic acids can “cloak” the vector from inducing unwanted immune responses in multiple, but not all, models.”

Immune responses in the subretinal space have different timing and effects than those in other tissues.

“suggesting that kinetics and impact of immune responses in the subretinal space are different than those in other tissues.”

scAAV8.FIX and scAAV8.FIX.io1 do not differ inherently in potency.

“suggesting that scAAV8.FIX and scAAV8.FIX.io1 do not have inherently different potencies.”

These results are consistent with adaptive T cell responses in the eye.

“These results are consistent with adaptive T cell responses in the eye.”

Clinically, the TLR9-inhibitory strategy may not fully suppress all immune responses, especially depending on dose, route, and transgene.

“our approach may not translate to complete suppression of all immune responses clinically, depending particularly on dose, route of administration and type of transgene.”

Short TLR9i sequences kept their immune-inhibitory function when placed in the much longer AAV vector genome.

“Our results demonstrated that the immune-inhibitory activity of short TLR9i sequences remained functional in a much longer strand of DNA, the AAV vector genome.”

In the mouse liver studies, plasma from PBS-injected mice produced signals comparable to a blank control, which demonstrated that the human factor IX ELISA kit was specific for human factor IX.

“Plasma from PBS-injected mice gave signals similar to that of a blank control, demonstrating specificity of the kit for human factor IX.”

Multiple ocular AAV gene therapy trials have reported intraocular inflammation at doses used therapeutically.

“Furthermore, several ocular AAV gene therapy trials have reported intraocular inflammation at therapeutically relevant doses (see Figure S18 and S19 for analysis).”

• cites Figure S18 and S19Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The intravitreal NHP experiment had substantial intra-group variability in inflammation and expression.

“Limitations of the intravitreal NHP experiment include a single experiment of small and underpowered sample size per group to demonstrate a clinically significant effect, substantial intra-group variability in inflammation and expression, and a lack of understanding of inflammation at the tissue level via histopathology or cell immune phenotype characterization.”

The intravitreal NHP experiment did not characterize tissue-level inflammation by histopathology or immune cell phenotyping.

“Limitations of the intravitreal NHP experiment include a single experiment of small and underpowered sample size per group to demonstrate a clinically significant effect, substantial intra-group variability in inflammation and expression, and a lack of understanding of inflammation at the tissue level via histopathology or cell immune phenotype characterization.”

In the highly immunogenic intravitreal NHP model, factors besides TLR9 may also drive intraocular inflammation.

“However, they also highlight that other immune factors in addition to TLR9 might contribute to intraocular inflammation in this highly immunogenic route of administration and model.”