Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism

Probably not a substantial claim: may not pass S3 (load-bearing).

Pervasive neuronal tropism extends to numerous shuffled AAV capsid chimeras that were recovered in a previous study.

“Similarly, this pervasive neuronal tropism also extends to numerous shuffled AAV capsid chimeras that were recovered in a previous study.17”

• cites [17] Gray SJ, Blake BL, Criswell HE, Nicolson SC, Samulski RJ, McCown TJ et al. Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB). Mol Ther 2010; 18: 570–578. sourcePartly supported

  The Results section on direct brain infusion reports that four recovered chimeric clones transduced cells, most of which resembled neurons. But it does not establish pervasive neuronal tropism across numerous recovered chimeras: one tested clone showed no transduction, and after intravenous administration only clones 32 and 83 transduced brain cells in the seizure-affected regions. No page numbers were available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

AAV9 can cross the blood-brain barrier and transduce both neurons and astrocytes after intravenous administration.

“AAV9 can cross the blood–brain barrier and transduce both neurons and astrocytes after intravenous administration.7,10–11”

• cites [7] Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Ther 2004; 10: 302–317. sourceNot checked — no full text of the source
• cites [10] Klein RL, Dayton RD, Leidenheimer NJ, Jansen K, Golde TE, Zweig RM . Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. Mol Ther 2006; 13: 517–527. sourceNot checked — no full text of the source
• cites [11] Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK . Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 2009; 27: 59–65. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

AAV vectors support long-term, non-toxic gene expression for up to 8 years in non-human primates.

“AAV vectors support long-term, non-toxic gene expression for up to 8 years in non-human primates.1–3”

• cites [1] Hadaczek P, Eberling JL, Pivirotto P, Bringas J, Forsayeth J, Bankiewicz KS . Eight years of clinical improvement in MPTP-lesioned primates after gene therapy with AAV2-hAADC. Mol Ther 2010; 18: 1458–1461. sourceNot checked — no full text of the source
• cites [2] Kantor B, McCown T, Leone P, Gray SJ . Clinical applications involving CNS gene transfer. Adv Genet 2014; 87: 71–124. sourcePartly supported

  The Introduction (§3.1; page unavailable) says AAV can provide long-term stable expression without associated inflammation or toxicity. The Parkinson disease section (§3.4; page unavailable) reports therapeutic effects from vector-derived AADC expression in primates up to eight years after transduction, but does not establish non-toxicity over that period in those animals. The immune-responses section (§4.1; page unavailable) also reports severe adverse effects in primates given an AAV9/GFP vector, so the safety claim needs qualification. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [3] Kantor B, Bailey RM, Wimberly K, Kalburgi SN, Gray SJ . Methods for gene transfer to the central nervous system. Adv Genet 2014; 87: 125–197. sourcePartly supported

  Section 2.2 (page not provided) reports AAV-delivered expression lasting at least 6 years in primates; it assigns the 8-year figure to dogs, not primates. The same section describes AAV vectors as having no known associated pathologies and causing a very mild immune response, which supports the safety characterization but does not establish an eight-year non-toxic outcome in primates. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

AAV8 and AAV5 transduce a significantly greater area of the CNS in comparison with AAV2.

“AAV8 and AAV5 transduce a significantly greater area of the CNS in comparison with AAV2”

• cites [7] Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol Ther 2004; 10: 302–317. sourceNot checked — no full text of the source
• cites [10] Klein RL, Dayton RD, Leidenheimer NJ, Jansen K, Golde TE, Zweig RM . Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. Mol Ther 2006; 13: 517–527. sourceNot checked — no full text of the source
• cites [11] Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK . Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 2009; 27: 59–65. sourceCould not verify

  The Introduction (page unavailable) says AAV8 showed poor CNS penetration after vascular delivery, but it does not compare AAV8 with AAV2. Searches for AAV5, AAV2, and CNS transduction area found no passage comparing the three serotypes or reporting a significant difference. (LLM, conf=0.77)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

These latter processes involve VP1- and VP2-specific portions of the capsid that contain acidification-responsive phospholipase domains and the nuclear localization signal.

“These latter processes involve VP1- and VP2-specific portions of the capsid that contain acidification-responsive phospholipase domains and the nuclear localization signal.20”

• cites [20] Nonnenmacher M, Weber T . Intracellular transport of recombinant adeno-associated virus vectors. Gene Therapy 2012; 19: 649–658. sourcePartly supported

  The Post-Endocytic Trafficking section identifies a phospholipase A2 domain in VP1 and describes exposure of the VP1 and VP2 N-termini during endosomal processing. The Nuclear Import section identifies nuclear-localization signals shared by VP1 and VP2. However, the paper does not place phospholipase domains in both proteins or describe those domains as acidification-responsive; it says acidic pH alone does not expose the VP1/VP2 N-termini. Page numbers were not available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

AAV capsids have nine variable regions surrounded by conserved regions where the variable regions form loops and clusters on the exterior of the capsid.

“The AAV capsids have nine variable regions surrounded by conserved regions where the variable regions form loops and clusters on the exterior of the capsid.18–19”

• cites [18] Agbandje-McKenna M, Kleinschmidt J . AAV capsid structure and cell interactions. Methods Mol Biol 2011; 807: 47–92. sourceNot checked — no full text of the source
• cites [19] Govindasamy L, Padron E, McKenna R, Muzyczka N, Kaludov N, Chiorini JA et al. Structurally mapping the diverse phenotype of adeno-associated virus serotype 4. J Virol 2006; 80: 11556–11570. sourcePartly supported

  The Results identify nine variable regions (I–IX) in a structural comparison of AAV4 and AAV2; they lie close to or on the capsid surface and cluster into surface features, while core structural elements are conserved. The claim omits that the count of nine comes from comparing these two serotypes, presenting it instead as a general property of AAV capsids. Our copy provides no page numbers for these passages. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

6-OHDA causes dopamine containing nerve terminals to degenerate, which leads to oligodendrocyte precursor cell infiltration.

“6-OHDA causes dopamine containing nerve terminals to degenerate, which leads to oligodendrocyte precursor cell infiltration.24,25”

• cites [24] Hughes EG, Kang SH, Fukaya M, Bergles DE . Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain. Nat Neurosci 2013; 16: 668–676. sourceCould not verify

  The Introduction (page unavailable) says NG2+ cell proliferation increases in chronic neurodegenerative disease, but does not address 6-OHDA, degeneration of dopamine-containing terminals, or precursor-cell infiltration after that degeneration. Searches for 6-OHDA and dopaminergic terminal degeneration found no passage establishing the claimed sequence. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [25] Kang SH, Li Y, Fukaya M, Lorenzini I, Cleveland DW, Ostrow LW et al. Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis. Nat Neurosci 2013; 16: 571–579. sourceCould not verify

  The Results section on degeneration of early-born oligodendrocytes examines motor-neuron loss after ricin injection, not dopamine-terminal degeneration after 6-OHDA. Our copy reports no 6-OHDA experiment or finding that loss of dopamine-containing terminals causes oligodendrocyte precursor infiltration (searched: 6-OHDA, 6-hydroxydopamine, dopaminergic axons, NG2 cells). (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Upon nuclear entry, the AAV virion uncoats, followed by AAV genome second-strand synthesis and, finally, transgene expression.

“Upon nuclear entry, the AAV virion uncoats, followed by AAV genome second-strand synthesis and, finally, transgene expression.20”

• cites [20] Nonnenmacher M, Weber T . Intracellular transport of recombinant adeno-associated virus vectors. Gene Therapy 2012; 19: 649–658. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

After cellular uptake into endosomes, AAV escapes from the endosome/lysosome and traffics to the perinuclear compartment.

“After cellular uptake into endosomes, AAV subsequently escapes from the endosome/lysosome and traffics to the perinuclear compartment.20”

• cites [20] Nonnenmacher M, Weber T . Intracellular transport of recombinant adeno-associated virus vectors. Gene Therapy 2012; 19: 649–658. sourcePartly supported

  The AAV ENDOCYTOSIS and POST-ENDOCYTIC TRAFFICKING sections support uptake by endocytosis, subsequent escape into the cytoplasm, and accumulation of many particles in a perinuclear compartment. They do not establish the claimed order of escape *before* trafficking to that compartment; the latter section says AAV2 and AAV5 appear to accumulate in the Golgi before release into the cytoplasm. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

AAV8 and AAV9 exhibit a dominant neuronal tropism after direct injection into the rodent striatum as well as in other brain structures.

“AAV8 and AAV9 exhibit a dominant neuronal tropism after direct injection into the rodent striatum9,13,16,27 as well as in other brain structures.12,28,29”

• cites [9] Harding TC, Dickinson PJ, Roberts BN, Yendluri S, Gonzalez-Edick M, Lecouteur RA et al. Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8-pseudotyped vectors. Hum Gene Ther 2006; 17: 807–820. sourceNot checked — no full text of the source
• cites [13] Cearley CN, Vandenberghe LH, Parente MK, Carnish ER, Wilson JM, Wolfe JH . Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain. Mol Ther 2008; 16: 1710–1718. sourcePartly supported

  The Abstract (page not provided) reports that AAV9 was injected into the adult mouse striatum and other brain structures and transduced neurons; the Discussion says AAV vectors generally transduce neurons preferentially. But this paper did not test AAV8 by direct injection into those adult brain structures, so it does not establish the claimed striatal tropism for AAV8. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [16] Lawlor PA, Bland RJ, Mouravlev A, Young D, During MJ . Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 2009; 17: 1692–1702. sourceNot checked — no full text of the source
• cites [27] Sondhi D, Hackett NR, Peterson DA, Stratton J, Baad M, Travis KM et al. Enhanced survival of the LINCL mouse following CLN2 gene transfer using the rh.10 rhesus macaque-derived adeno-associated virus vector. Mol Ther 2007; 15: 481–491. sourceNot checked — no full text of the source
• cites [12] Cearley CN, Wolfe JH . Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol Ther 2006; 13: 528–537. sourceNot checked — no full text of the source
• cites [28] Taymans JM, Vandenberghe LH, Haute CV, Thiry I, Deroose CM, Mortelmans L et al. Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum Gene Ther 2007; 18: 195–206. sourceNot checked — no full text of the source
• cites [29] Klein RL, Dayton RD, Tatom JB, Henderson KM, Henning PP . AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method. Mol Ther 2008; 16: 89–96. sourceNot checked — no full text of the source

May combine two claims.

GFP-positive cells exhibited the typical oligodendrocyte morphology with clear labeling of myelin in the patch component of the striatal patch/matrix.

“GFP-positive cells exhibited the typical oligodendrocyte morphology with clear labeling of myelin in the patch component of the striatal patch/matrix (Figure 1b).”

May combine two claims.

The majority of the cells in the mixed glial cell cultures are oligodendrocytes and are present in a similar percentage between the AAV8 and Olig001 conditions.

“indicating that the majority of the cells are oligodendrocytes and present in a similar percentage between the two conditions.”

May combine two claims.

The AAV8-treated sister plate contained 89% O4-positive cells and the Olig001-treated sister plate contained 86% O4-positive cells.

“The AAV8-treated sister plate contained 89% O4-positive cells and the Olig001-treated sister plate contained 86% O4-positive cells”

May combine two claims.

When Olig001 was administered intravenously to unilateral 6-OHDA-treated rats, 2 weeks later immunohistochemistry revealed only a few GFP-positive neurons in the ipsilateral striatum and a sparse number of oligodendrocyte-like cells.

“When the chimeric virus (named Olig001) was administered intravenously to unilateral 6-OHDA-treated rats, 2 weeks later immunohistochemistry revealed only a few green fluorescent protein (GFP)-positive neurons in the ipsilateral striatum and a sparse number of oligodendrocyte-like cells (data not shown).”

May combine two claims.

GFP-positive cells exhibit the typical morphology of striatal oligodendrocytes and co-localize with two oligodendrocyte markers, Olig2 and myelin basic protein.

“GFP-positive cells exhibit the typical morphology of striatal oligodendrocytes and co-localize with two oligodendrocyte markers, Olig2 (Figure 1e–g) and myelin basic protein (Figure 1h–j).”

May combine two claims.

Almost all of the GFP-positive cells (>95%) were oligodendrocytes with only a few neurons and no GFP-positive astrocytes or microglia.

“Thus, almost all of the GFP-positive cells (> 95%) were oligodendrocytes with only a few neurons and no GFP-positive astrocytes or microglia.”

Classified as probably not a substantial claim.

Some AAV capsids exhibit a weak tropism for oligodendrocytes when a cell-specific promoter drives expression (myelin basic protein).

“Some AAV capsids exhibit a weak tropism for oligodendrocytes when a cell-specific promoter drives expression (myelin basic protein).”

• cites [15] Chen H, McCarty DM, Bruce AT, Suzuki K, Suzuki K . Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus. Gene Therapy 1998; 5: 50–58. sourceNot checked — no full text of the source
• cites [16] Lawlor PA, Bland RJ, Mouravlev A, Young D, During MJ . Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 2009; 17: 1692–1702. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

No serious adverse effects have been attributed to the AAV vectors to date.

“yet to date, no serious adverse effects have been attributed to the AAV vectors.4–6”

• cites [4] McPhee SW, Janson CG, Li C, Samulski RJ, Camp AS, Francis J et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med 2006; 8: 577–588. sourceNot checked — no full text of the source
• cites [5] Muramatsu S, Fujimoto K, Kato S, Mizukami H, Asari S, Ikeguchi K et al. A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson’s disease. Mol Ther 2010; 18: 1731–1735. sourceNot checked — no full text of the source
• cites [6] Rafii MS, Baumann TL, Bakay RA, Ostrove JM, Siffert J, Fleisher AS et al. A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer’s disease. Alzheimers Dement 2014; 10: 571–581. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The only exception is AAV4, which transduces ependymal cells, but not neurons, astrocytes, microglia or oligodendrocytes.

“The only exception is AAV4, which transduces ependymal cells, but not neurons, astrocytes, microglia or oligodendrocytes.8”

• cites [8] Davidson BL, Stein CS, Heth JA, Martins I, Kotin RM, Derksen TA et al. Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc Natl Acad Sci USA 2000; 97: 3428–3432. sourceNot checked — no full text of the source

May combine two claims.

These studies generated a novel chimeric AAV capsid variant with a preferential in vivo tropism for oligodendrocytes, a finding that represents a major departure from the normal neuronal tropism indicative of the parental AAV serotypes, except AAV4.

“These studies generated a novel chimeric AAV capsid variant with a preferential in vivo tropism for oligodendrocytes, a finding that represents a major departure from the normal neuronal tropism indicative of the parental AAV serotypes, except AAV4.”

May combine two claims.

Past studies have described the ability of AAV2 or AAV8 to transduce oligodendrocytes at a low efficiency, but these studies required the use of oligodendrocyte-specific promoters to prevent expression in neurons.

“Past studies have described the ability of AAV2 or AAV8 to transduce oligodendrocytes at a low efficiency, but these studies required the use of oligodendrocyte-specific promoters to prevent expression in neurons, the preferred cell type for these vectors.15,16”

• cites [15] Chen H, McCarty DM, Bruce AT, Suzuki K, Suzuki K . Gene transfer and expression in oligodendrocytes under the control of myelin basic protein transcriptional control region mediated by adeno-associated virus. Gene Therapy 1998; 5: 50–58. sourceNot checked — no full text of the source
• cites [16] Lawlor PA, Bland RJ, Mouravlev A, Young D, During MJ . Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 2009; 17: 1692–1702. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S2 (checkable).

The ability to efficiently target oligodendrocytes in vivo could advance therapeutic strategies for demyelinating diseases such as Canavan disease or Krabbe disease.

“Moreover, the ability to efficiently target oligodendrocytes in vivo could advance therapeutic strategies for demyelinating diseases such as Canavan disease or Krabbe disease.”

May combine two claims.

In the striatum, Olig001 exhibits a preferred tropism for oligodendrocytes and low tropism for neurons, which is distinct from any previously reported AAV capsid.

“Thus, in the striatum Olig001 exhibits a preferred tropism for oligodendrocytes and low tropism for neurons, which is distinct from any previously reported AAV capsid.”

Classified as probably not a substantial claim.

The first step of AAV transduction entails AAV binding to a primary receptor and co-receptor on the cell surface.

“The first step of AAV transduction entails AAV binding to a primary receptor and co-receptor on the cell surface.18”

• cites [18] Agbandje-McKenna M, Kleinschmidt J . AAV capsid structure and cell interactions. Methods Mol Biol 2011; 807: 47–92. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Outliers were determined using the Grubbs test and subsequently removed.

“Outliers were determined using the Grubbs test and subsequently removed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At least five independent fields were imaged and used for quantification of O4-positive cells in AAV8- and Olig001-treated plates.

“At least five independent fields were imaged on a Olympus IX71 inverted fluorescent microscope with cellSens Entry Software (Japan) and used for quantification of O4-positive cells in AAV8- and Olig001-treated plates.”

May combine two claims.

The novel AAV vector shows increased extracellular binding to oligodendrocytes in vitro, suggesting that the increased oligodendrocyte tropism is a result of external receptor interactions rather than downstream intracellular events.

“Further, it shows increased extracellular binding to oligodendrocytes in vitro suggesting that the increased oligodendrocyte tropism is a result of external receptor interactions rather than downstream intracellular events.”

May combine two claims.

The procedure of incubating mixed glial cell cultures with virus for 1 h at 4 °C allows vector binding to the cell surface, but prevents internalization.

“This procedure allows vector binding to the cell surface, but prevents internalization.31”

• cites [31] Xiao PJ, Li C, Neumann A, Samulski RJ . Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues. Mol Ther 2012; 20: 317–328. sourceNot checked — no full text of the source

May combine two claims.

The biodistribution of Olig001 indicates that it is not able to cross the blood-brain barrier, which was confirmed by a lack of GFP-positive staining in the brain by immunohistochemistry.

“The biodistribution of Olig001 indicates that it is not able to cross the blood–brain barrier, which was confirmed by a lack of GFP-positive staining in the brain by immunohistochemistry (data not shown).”

Classified as probably not a substantial claim.

The unique topology of the AAV capsid confers cellular tropism by differential receptor binding.

“The unique topology of the AAV capsid confers cellular tropism by differential receptor binding.”

• cites [18] Agbandje-McKenna M, Kleinschmidt J . AAV capsid structure and cell interactions. Methods Mol Biol 2011; 807: 47–92. sourceNot checked — no full text of the source

May combine two claims.

Our data describes a unique AAV vector with preferential oligodendrocyte tropism within the CNS and clear detargeting from peripheral tissues when intravenously injected.

“In total, our data describes a unique AAV vector with preferential oligodendrocyte tropism within the CNS and clear detargeting from peripheral tissues when intravenously injected.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures received prior approval by the University of North Carolina Institutional Animal Care and Use Committee.

“All care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures received prior approval by the University of North Carolina Institutional Animal Care and Use Committee.”