This weekend, long-term follow-up data from two AAV gene therapies – a vector from University of Pennsylvania (UPenn) and a vector from University College of London (UCL) – used to treat patients with Leber’s congenital amaurosis (LCA) were published in The New England Journal of Medicine (NEJM) [1,2]. In summary, both of these studies demonstrated that AAV2 delivery of RPE65 resulted in an initial improvement in vision, which then began to decrease after 1-3 years. This made waves in the investment community as investors concluded that AAV gene therapies may not be long lasting in the eye, which has a significant read through to Spark’s SPK-RPE65 and other AAV gene therapy companies.
Spark opened down over 15% on Monday but bounced back as analysts and Spark management clarified some key differences between these vectors’ design, delivery, and manufacturing. Based on available data I tend to agree – that the concerns are overblown. Here I explain why the shortcomings of UPenn and UCL’s data are most likely inadequacies specific to these vectors’ design, delivery, and/or manufacturing; thus, read through to AAV gene therapies as a whole has little scientific basis.
First, some background on the vectors and LCA is required. A summary of the vector specifications, including Spark Therapeutics’ SPK-RPE65, are below:
Transgene: human RPE65 with Kozak sequence (with CMV enhancer)
Promoter: hybrid Beta-actin promoter
Delivery route: Subretinal injection
Manufacturing: Proprietary HEK293 triple transfection method with purification of product leading to essentially no empty capsids. In addition, vector product is mixed with surfactant to enable full delivery of vector product from needle
Dose: 1.5E11 | Volume: 300 µl
LCA resulting from RPE65-mutations is a 2-pronged disease that leads to loss in vision. It’s caused by the dysfunction and degeneration of photoreceptors. The hope for gene therapy in LCA is that the delivery and resulting production of functional RPE65 protein can correct the dysfunction and protect/reverse degeneration. I’ll focus on the UPenn results since these data seem to have received the most attention this week.
Primary conclusions from UPenn’s recent publication:
Three years after receiving the therapy, improvement in vision was maintained in the treated eye; however, there was a continual loss of photoreceptors
Areas of improved visual sensitivity expanded slowly over a period of 1-3 years in treated eyes, but the area of improvement subsequently declined
These two conclusions in tandem suggest that although there is successful transfection and production of RPE65, gene therapy for LCA using UPenn’s current vector system is unable to ‘cure’ this disease. I see two possible reasons:
UPenn’s AAV vector/delivery system is inadequate for proper magnitude and widespread expression of RPE65 in the eye, which would not facilitate a ‘cure’. Further, the AAV vector may be immunogenic, leading to eventual vector/transgene destruction. If this is the case, AAV gene therapy delivering RPE65 could lead to a cure for LCA (correction of both features of the disease) with further vector/delivery optimization.
Delivery of the RPE65 gene using AAV vectors – even with adequate magnitude and widespread expression – may not correct both aspects of LCA. Essentially, correcting RPE65 alone may not be enough (underlying biology).
Surprisingly, UPenn drew a similar conclusion in a previous publication of earlier follow-up data from the same clinical trial . The investigators concluded in 2013 that, “all three analysis methods supported the conclusion that gene therapy has not modified the natural history of progressive retinal degeneration in the RPE65-LCA patients” .
The progressive retinal degeneration – as discussed above – is one half of the two-pronged nature of LCA. Retinal degeneration is commonly measured by comparing the ONL fraction between treated, untreated, and natural history studies. As shown below, progressive thinning of the ONL for both the untreated eye (left) and treated eye (right) are indistinguishable from each other and the natural history data .
These data suggests that UPenn’s AAV vector delivering RPE65 corrects only the dysfunction of photoreceptors, and not their degeneration. This is further supported by the improvement in visual sensitivity (and subsequent decline) for treated eyes as ONL fraction continues to thin – indicative of photoreceptor degeneration :
Referencing only UPenn’s data, it’s unclear what’s responsible for the transient nature of UPenn’s therapy. I’m tempted to conclude it’s the underlying biology, but after analyzing UCL and Spark’s data, I believe it could also be a result of inadequacies in the vector.
As mentioned, UCL also published long-term follow-up data for their RPE65 AAV gene therapy, tgAAG76, this weekend. The investigators conclude, “Improvements in retina sensitivity were evident, to various extents, in six [out of twelve] participants for up to 3 years, peaking at 6 to 12 months and then declining” . In addition, five of the eight participants in the high dose cohort had intraocular inflammation or immune responses.
I believe the contributing factors responsible for the shortcomings in tgAAG76’s data are inadequacies in their vector design, delivery, and manufacturing. First, tgAAG76 employs a partial human RPE65 promoter, which despite having benefits for restricting transgene expression, might not be adequate to drive enough transgene expression. This may be why UCL used a higher dose compared to SPK-RPE65 and UCL. Second, subretinal injection for the delivery of tgAAG76 resulted in a relatively long recovery period for the eye – something not seen in other AAV RPE65 gene therapies. Sparing details, this is most likely because of differences in the subretinal injection methodology along with the dosing volume used. In addition, UCL used an “ancient” manufacturing method for the tgAAG76 AAVs – infecting the B50 cell line with a helper Adenovirus. This older approach theoretically leads to contaminants in the final product, creating the risk of an immune response. Newer manufacturing method have generated more consistent products.
Bottom line – it’s not surprising to see tgAAG76’s efficacy wane with time. From the ground up there are inadequacies.
These data in tandem with the UPenn data seem to suggest that AAV delivery of RPE65 may not be adequate to cure LCA, however referencing only these two data packages the reason is unclear. Possibilities include:
Inadequate vectors prevent durable and widespread RPE65 expression. Proper expression could theoretically correct both aspects of LCA.
Underlying biology: Delivery of RPE65 simply will not prevent further degradation of photoreceptors and the resulting loss of vision.
AAV transgene expression wanes over time, possibly because this is a non-integrating transgene.
I feel comfortable, with available data, ruling out reason 3. There’s a plethora of data demonstrating long-term expression of AAV transgenes in both animals and humans. For instance, at least 7 years in RPE65-deficient Braird dogs . Spark has data demonstrating transgene expression for at least 5 years from their SPK-RPE65 clinical trials, and transgene Factor IX expression for at least 10 years after intramuscular injection in another clinical trial [4,5].
With SPK-RPE65’s current data package, I’m comfortable concluding that it’s inadequacies in UPenn and UCL’s vector that are a key contributor for the shortcomings in their long-term data. However, I can’t rule out the possible contribution of the underlying biology – that once a threshold of photoreceptor degeneration has been reached, continuing degradation cannot be prevented by RPE65 gene therapy.
Key differences in SPK-RPE65 compared to UPenn and UCL's vector:
Different immunosuppressive regimen
Different transgene cassette: “optimized” (Kozak sequence) human RPE65 transgene
Different manufacturing process – Spark’s HEK293 triple transfection incorporates purification that removes a substantial amount of empty capsids.
Different subretinal injection
SPK-RPE65 gene therapy formulation includes mixing with a surfactant to prevent clumping of AAVs on the inside of the needle (shown below). Clumping leads to lower amounts of of delivered AAV vector from the needle to the eye – which Spark (CHOP) identified prior to initiating SPK-RPE65 clinical trials. This is shown graphically below.
Clearly, there are multiple modest difference between SPK-RPE65 and the vectors used by UPenn and UCL. Despite their modesty, these may lead to significant differences in vector performance in preclinical testing and in long-term clinical outcomes for SPK-RPE65 treated patients compared to UPenn/UCL vector treated patients.
The current SPK-RPE65 data package contrasts data from UPenn/UCL because SPK-RPE65 treated patients apparently do not demonstrate the same decrease in vision as patients who received the above two AAV RPE65 gene therapies. Spark stated in their earnings release on May 6, “SPK-RPE65 continues to demonstrate long-lasting effects, with subjects reported to date from [Spark’s] Phase I study of the contralateral eye maintaining improvements in function, vision, and retinal sensitivity through their latest follow-up visit, which ranges from two to four years post-injection” . This supports my view that SPK-RPE65 may be able to have an effect on both photoreceptor dysfunction and degeneration, but without further data, I cannot be 100% confident.
SPK-RPE65’s positive clinical outcomes could theoretically wane over time as treated subjects age – due to the underlying photoreceptor degeneration which may still be occurring. Even if this is the case, this would be a RPE65 therapy-specific issue (no read through to AAV gene therapies as a whole), and it would still be a meaningful outcome. In her blog this morning, Dr. Ricki Lewis does a fantastic job of explaining how even if RPE65 gene therapy is transient in nature, it is still very meaningful for patients diagnosed with LCA .
The Big Picture
In conclusion, a negative read through from the UPenn/UCL data to AAV gene therapies as a whole lacks scientific basis. To re-emphasize, the above comparisons of the three most advanced AAV RPE65 gene therapies is also meant to highlight the importance of all aspects of an AAV gene therapy: design, delivery, and manufacturing. This can also be seen in analyzing previous literature concerning AAV gene therapy for hemophilia B. For example, there are distinct differences in efficacy for AAV2 hemophilia trial being delivered via intramuscular injection and delivery via peripheral vein injection for liver transfection [8,9]. In addition, there are clear differences in magnitude and duration of liver-mediated expression of FIX for AAV2 vectors versus AAV8 vectors .
Last, it’s worth mentioning that certain diseases are easier to treat via gene therapy due to their monogenic nature (example: hemophilia) whereas complex diseases such as heart failure – a non-monogenic disease – is much more difficult to treat via gene therapy. The nature of the disease being treated must be taken into consideration when setting expectations for gene therapies.
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